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Chiroptical spectroscopy of nitrogenase

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Chiroptical spectroscopy of nitrogenase

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Content CHIROPTICAL SPECTROSCOPY OF NITROGENASE by Hop Trung Nguyen A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial F ulfillm ent of the Requirements for the Degree DOCTOR OF PHILOSOPHY (Chemistry) March 1981 UNIVERSITY OF SOUTHERN CALIFORNIA TH E GRA DU ATE SC HO O L U N IV E R S IT Y PARK LOS A NG ELES. C A L IF O R N IA 9 0 0 0 7 This dissertation, written by H 0P _JR yN 6_N G U Y E N ......... under the direction of h.J$... Dissertation Com mittee, and approved by all its members, has been presented to and accepted by The Graduate School, in partial fulfillment of requirements of the degree of D O C T O R O F P H I L O S O P H Y PA • T > . C ' 81 j\ j y j/ DISSERTATION COMMITTEE sAAii A V rtv T . Chairman To my parents To my brothers and s is te r s ACKNOW LEDGEMENTS I am deeply grateful to my parents for th e ir continuous support and encouragement throughout my college and graduate studies. I value the guidance and appreciate the financial support (research assistantship) given by Professor Charles E. McKenna during my graduate research. I would also like to thank Professor P h ilip J. Stephens for the use of the CD and MCD instruments in his laboratory. I would like to acknowledge the contributions from the following colleagues: Candice Huang, M arie-Claire McKenna, Jay Jones, Harutyun Eran, and Alan Osumi (protein p u rific a tio n ), Carsten Kampe (metal analysis), Frank Devlin (CD and MCD measurements), and Vance Morgan (EPR measurements). I would also like to thank my brother Hai Nguyen for the technical illu s tra tio n s in this dissertation, and M arie-Joelle Tran Huu for her typing. i i i TABLE O F CONTENTS Page DEDICATION.............................................................................................................................. ii ACKNOWLEDGEMENTS......................................................................................................................i ii LIST OF TABLES....................................................................................................................... v i i i LIST OF FIGURES ............................................................................................. ix CHAPTER 1 : GENERAL INTRODUCTION........................................................................... 1 1.1. Nitrogenase................................................................................................... 1 1.2. Iron-Sulfur Clusters ............................................................................. 3 1.3. CD Spectroscopy of Iron-Sulfur Proteins.................................. 5 1.4. Magnetic Circular Dichroism........................................... 7 1.5. CD and MCD of N2 ase Components..................................................... 8 CHAPTER 2: MULT I GRAM PURIFICATION OF AZOTOBACTER VINELAND I I NITROGENASE PROTEINS........................................................................... 13 2 .1. In tro d u c tio n ............................................................................................... 13 2.2. Equipment. . .......................................................................................... 14 2.3. Chemicals........................................................................................................ 16 2.4. Cel I Breaking............................................................................................... 17 2 .5 . Protamine Sulfate P re c ip ita tio n ..................................................... 18 2 .6. F irs t DEAE Column..................................................................................... 19 i v Page 2 .7 . Further P u rification of FeMo Component .................................. 21 2 .7 .1 . Heat Step and Second DEAE Column . . . . . . . 21 2 .7 .2 . C rystallizatio n of FeMo Component.............................. 23 2 .8. Further Pu rifica tio n of Fe Component................................ 24 2 .8 .1 . Linear Gradient Chromatography .................................. 24 2 .8 .2 . Sephadex G-100 Chromatography....................................... 25 2 .9. Purity Analyses.......................................................................................... 26 2 .9 .1 . Introduction ............................................................................. 26 2 .9 .2 . ^ 2 ^ 2 Eduction Assays......................................................... 27 2 .9 .3 . Biuret Analysis....................................................................... 28 2 .9 .4 . Gel EIectrophoresis.............................................................. 28 2 .9 .5 . Metal Analysis ....................................................................... 30 2 .9 .6 . Detection of Heme Impurities by MCD Spectroscopy ........................................................................... 31 2.10. Conclusion.................................................................................................. 32 CHAPTER 3: CD AND MCD OF NITROGENASE PROTEINS.......................................... 61 3 .1 . In tro d u c tio n ............................................................................................... 61 3.2. Chemicals....................................................................................................... 62 3.3. R eagents........................................................................................................ 62 3 .4 . Equipment........................................................................................................ 63 3.5. Protein Sample Preparations.............................................................. 64 3.6. Results and D iscu ssio n....................................................................... 65 3 .6 .1 . FeMo Proteins............................................................................ 65 3 .6 .2 . Fe Proteins................................................................................ 67 v Page 3.7. Conclusion................................................................................................... 69 CHAPTER 4: MEDIUM AND TEMPERATURE EFFECTS ON REDUCED AV2 . . . . 76 4 .1. In tro d u c tio n ............................................................................................... 76 4 .2 . E xp erim en tal............................................................................................... 76 4 .2 .1 . General Preparations of Protein Samples. . . . 76 4 .2 .2 . Variation of Ion ic- Strength........................................... 77 4 .2 .3 . Variation of pH...................................................................... 78 4 .2 .4 . Variation of B u ffer............................................................. 78 4 .2 .5 . Variation of Temperature ............................................... 78 4 .2 .6 . Spectroscopic Measurements .................................. .... . 79 4.3. Results and D iscu ssio n....................................................................... 79 CHAPTER 5: FE ION CHELATION AND CD STUDIES OF AV2 PROTEIN. . . . 89 5.1. In tro d u c tio n .............................................................. 89 5 .2. E xp erim en tal............................................................................................... 90 5 .2 .1 . Controlled 0 ^~Inactivat ion of Av2................... 90 5 .2 .2 . Chelation of Fe Ions from Av2 by B P D.......... 91 5 .2 .3 . CD of 0 ^ - I nactivated A v 2 ..................................... 93 5 .3. Results and D isc u s s io n....................................................................... 93 CHAPTER 6 : AV2 "SELF-OXIDATION11................................................................................ 104 6 .1 . In tro d u c tio n .....................................................................................................104 6 .2 . E xperim en tal.................................................................................................... 105 6 .3 . Fe Protein "Self-O xidation"....................................................................107 v i Page 6 .4 . The Decomposition of D ith io n ite in "Self-Oxidized” Av2.................................................................................................................... 109 6 .5 . S p ecificity of MgATP for "Self-Oxidized" Av2...................... 110 6 .6 . EPR of "Self-Oxidized" A v 2 ..................................................................111 6 .7 . C onclusion.......................................................................................................112 CHAPTER 7: ATP AND ADP TITRATIONS OF DYE-OXIDIZED AV2 CD SPECTRA.......................................................................................................130 7.1. In tro d u c tio n ...................................................................................................130 7 .2 . E x p erim en tal..................................................................................................132 7 .3. ATP and ADP Bind Oxidized Av2.............................................................135 7 .4 . Difference Between the Bindings of ATP and ADP to Ox i d i zed A v 2 ............................................................................................. 136 7 .5. Stoichiometries and Dissociation Constants of the Bindings of ATP and ADP to Oxidized Av2.......................................137 REFERENCES................................................... 155 v i i LIST O F TABLES Page 2-1. P u rification of AVOP Nitrogenase.............................................................. 34 2-2. P u rification of FeMo Component. .......................................................... 35 2-3. P u rificatio n of Fe Component....................................................................... 36 2-4. Protocol for Acetylene Assays ................................................................... 37 2-5. P u rification of FeMo and Fe Proteins from 2 Kg of Azotobacter v in e la n d ii.................................................................................... 38 2-6. Mo and Fe Compositions of Av1 and Av2 Proteins................................ 40 5-1. BPD Chelation of Fe Ions from A v 2 .......................................................... 95 v i I i LIST O F FIGURES Page 1-1. Structures of 1-Fe, 2-Fe, 4-Fe, 3-Fe Iron-Sulfur Clusters . 11 2-1. Plot of Units of ^ 2 ^ 2 ^c+ivi+Y as a Function of Added PS-R.............................................................................................................................. 41 2-2. Experimental Set-Up of the F ir s t DEAE-CelIulose Column Chromatography....................................................................................................... 43 2-3. Elution P ro file of the F irs t DEAE-CeI IuIose Column.................. 45 2-4. Experimental Set-Up of the Heat-Step on FeMo Component. . . 47 2-5. Elution P ro file of the Second DEAE-CelIulose Column . . . . 49 2-6. Elution P ro file of the Linear-Gradient Column on Fe Component................................................................................................................ 51 2-7. Elution P ro file of the Sephadex G-100 Column................................ 53 2-8. Anaerobic Disc-Electrophoresis Patterns of Purified FeMo and Fe Fractions.................................................................................................. 55 2-9. V isible MCD Spectrum of Semi-Purified A v 1 ..................................... 57 2-10. V isible MCD Spectrum of C rystalline A v 1 ......................................... 59 3-1. Diagram of the Gas-Tight Sample Cell Holder (Medial Section and Front View) ................................................................................ 70 3-2. Absorption ( a ), CD (b ), and MCD (a t 1 Tesla) (c) Spectra of Reduced Av1 (------ ) and Kpl (----) ............................................................ 72 3-3. Absorption ( a ), CD (b ), and MCD (a t 1 Tesla) (c) Spectra of Reduced Av2 (------), Reduced Kp2 (— — - ) , and Post-Steady-State Oxidized Av2 (-----) ..................................................... 74 ix Page 4-1 . CD AR Spectra of Reduced Av2 as a Function of NaCI Concentration .................................................................................................. 4-2. CD AR Spectra of Reduced Av2 as a Function of pH................... 83 4-3. CD AR Spectra of Reduced Av2 as a Function of Buffer. . . 85 4-4. CD AR Spectra of Reduced Av2 as a Function of Temperature 87 5-1 . Time Course of Av2 A ir-1nactivat i o n ............................................... 96 5-2. Valve Positions during the Spectroscopic Studies of BPD Fe Chelation of Av2 .................................................................................... 5-3. Fe Equivalents Chelated by BPD in the Presence of MgATP as a Function of Av2 Specific A c tiv ity .......................................... . 100 5-4. Plots of e, Ae, and Ae/s as a Function of Av2 SA................... . 102 6 -1 . Visible-near UV Absorption (a) and CD (b) Spectra of Reduced (------), Av1-0xidized ( - • - • - ) , and "Self-Oxidized" Av2 (-----) ............................................................................................................ 6 - 2 . Visible-near UV CD Spectra of Reduced (------) and "Self-Oxidized" Av2 ( -----) in the Presence of MgATP. . . . . 116 6-3. Visible-near UV CD Spectra of Reduced Av2 in the Presence of MgAMP (-----), MgGTP ( ), MgCTP (----------), and MgUTP ( ..........) ................................................................................................................. 6-4. Visible-near UV CD Spectra of Reduced and "Self-Oxidized" Av2 in the Presence of MgAMP (-----), MgGTP (-------), MgCTP ( ---------), and MgUTP ( ...........) ........................................................................ 6-5. Visible-near UV CD Spectra of Reduced (------) and "Self-Oxidized" (-----) Av2 in the Presence of MgADP. . . . . 122 6 - 6 . Effects of "Self-Oxidation" on the EPR of Av2 ........................ X Page 6-7. Effects of MgATP on the EPR of Reduced and "Self-Oxidized" A v 2 .............................................................................................................................126 6- 8 . Effects of MgADP on the EPR of Reduced and "Self-Oxidized" A v 2 ..............................................................................................................................128 7-1. Front and Side View of Sample Cell Boat Used for Sequential T it r a t io n ..............................................................................................139 7-2. Visible-near UV CD Spectra of Dye-Oxidized Av2 (—— ), Dye-Ox i d i zed Av2 + MgATP (------ ), and Dye-Ox i d i zed Av2 + MgADP <--------) .............................................................. 141 7-3. T itra tio n of Dye-Oxidized Av2 CD with MgATP........................................ 143 7-4. T itra tio n of Dye-Oxidized Av2 CD with MgADP........................................ 145 7-5. Plots of % Change in CD at 360 nm as a Function of ATP/Av2 or ADP/Av2 Mole R a t i o ........................................................................................... 147 7-6. Plots of % Change in CD at 410 nm as a Function of ATP/Av2 or ADP/Av2 Mole R a t i o ........................................................................................... 149 7-7. Plots of % Change in CD at 470 nm as a Function of ATP/Av2 or ADP/Av2 Mole R a t i o ........................................................................................... 151 7-8. Effects of Av2 Specific A c tiv ity to the T itra tio n Curve at 360 n m ......................................................................................................................153 x i CHAPTER 1 GENERAL INTRODUCTION 1.1. Nitrogenase Nitrogenase ^ a s e ) is the enzyme responsible for nitrogen fixatio n in various nitrogen-fixing organisms^ and is currently the 2— 6 subject of intensive studies. Accounting for a ll natural nitrogen fix a tio n , the enzyme is functionally important to agronomy and the world food supply. N2 ase catalyzes the ATP hydrolysis-dependent reduction of nitrogen to ammonia in the presence of a suitable electron donor: N2 + 8 H+ + 8 a~ N2ase ^ 2NH^ + nATP nADP + nPI The active N^ase complex is composed of two metaIloproteins: the FeMo component and the Fe component. In the case of Azotobacter vine I and ? i , the Iarger component (referred in the Iite ra tu re as Av1, MoFe, or FeMo protein) has an estimated molecular weight (MW) of 220,000-270,000 daltons^ ^ and consists of 2 pairs non-identical 12 subunits. Av1 contains 1.5-2.0 Mo, 20-32 Fe, and 20-28 "acid 7-11 labile" sulfur atoms. The metal and su lfid e ions are believed to be arranged in protein bound clusters. The smaller component from A. vinelandi i (called Av2 or Fe protein) has an estimated M W of 1 60,000-68,000 daltons.^ ^ Av2 is dimeric ( i . e . 2 identical 7 8 = subunits), ' and believed to contain no Mo, 4 Fe, and 4 S bound 7-9 atoms per dimer. Both components are essential for the c a ta ly tic function of nitrogenase, and have the tendency to be irreversibly inactivated by 0 ^ in th e ir purified s t a t e s . ^ A cid ificatio n of FeMo protein in c i t r i c acid-phophate buffer followed by NMF washings, 14 results in the extraction of an iron-molybdenum cofactor or FeMo-co. FeMo-co is capable of restoring c a ta ly tic a c tiv ity to 14 15 nitrogenase-deficient extracts of Azotobacter mutant called UW45. ' The m etaI/suIfide stoichiometry was reported to be ™ 13 15 16 6 - 8 Fe:1 Mo: 6 S . ' ' The analysis of FeMo-co amino acid content 17 18 remains unsettled. 9 It is possible that FeMo-co exists as a metal cluster unattached to any peptide residues. Since neither isolated component is individually active, they are usually recombined during the in v itro determination of nitrogenase a c tiv ity . The a c tiv ity of each component is obtained from the t it r a t io n curves in which the amounts of substrate reduced are plotted as a function of increasing amounts of one component, while keeping 19 the other component constant. Saturation is reached at FeMorFe stoichiometries of 1:1 to 1:2. Sodium d ith io n ite is commonly used as electron donor in the a c tiv ity assays. Optimal conditions for A. vin elan d i? nitrogenase substrate reduction include a pH s lig h tly above 20 21 7, and a temperature between 20° and 40°C. ' The enzymic reduction of i^ is accompanied by the reduction of H^0+ to H2 « The ATP-dependent H2 evolution also occurs in the absence of reducible susbtrate. There are several available methods for 2 measuring nitrogenase a c tiv ity . They include the Nessler colorim etric 22 23 15 24 25 determination of NH^, ' the use of N2 , H2 analysis, and the 26 acetylene reduction method. Among these methods, the acetylene reduction method was found most simple and less time consuming. Acetylene is c a ta ly tic a lIy reduced to ethylene, and the gases can be analyzed by gas chromatography. H2 evolution is shut o ff during the production of ethylene. The a f f in it y for acetylene, measured by the 26 apparent Michealis constant Km of 0 .1 -0 .3 mM, is comparable to that of N2 (Km=0.06-0.12 mM) . 11 All N2 ase-catalyzed reactions necessite the hydrolysis of ATP to ADP and P .. Substrate reduction is believed to require 2 ATP per 26 electron transferred. Other substrate of nitrogenase include HN3 /N3- , 28 N2 0 , 29 RCeN, 2 8 , 3 0 RN=C, 3 1 - 3 4 RCeCH, CH2 =C=CH2 , 35 and 7,7 cyclopropene. 9 Cyclopropene has been used as chemical probe for 36*38 the topology and other aspects of the N2ase active s ite . CO is not reduced by N2 ase, but strongly inhibits all substrate reductions with the exception of H2 evolution. 2 8 , 3 0 1.2. Iron-Sulfur Clusters L i t t l e information is available about the structural organization of the Fe (and Mo) in the two nitrogenase components. In spite of a 39-44 .. 16 45-48 large body of spectroscopic studies (ESR, Mossbauer, ' 49 50 51 52 EXAFS, 9 and core extrusion methodologies ' ), the arrangements of the metal chromophores in N2ase remain to be elucidated. The presence of Fe and lab ile S atoms in the enzyme has suggested that 3 iron-sulfur cluster structures may be present. There are now several 53 we I I-characterized iron-sulfur proteins containing 1 , 2 , 4 , 8 , and 54 recently 3-Fe atoms. 55 The simplest Fe-S structure Is found In rubredoxlns. Fe is tetrahedraI Iy coordinated to four cysteine S atoms of the protein. The two oxidation states of rubredoxins are: hlgh-spin F e ( l l l ) , and hlgh-spin F e ( I I ) . The 2-Fe Iron-sulfur cluster Is found In plant, a lg a l, and mammalian ferrodoxins. The two Fe ions are bridged by two S” atoms. Each Fe is tetrahedral Iy bound to two sulfide atoms and to the protein 56 57 via two cysteine S atoms. * The reduction of the 2 Fe cluster Involves the formal valence change from Fe( I I I )— F e ( I I I ) to 56 57 F e ( I I I ) — Fe71 The visible-near UV spectra of iron-sulfur proteins resu lt from the Fe+S charge transfer excitation s. Thus, they are broad and have not been useful for theoretical interpretations. The extension to the neai— IR has been proven more p ro d u c tiv e .^ ^ The d-*d transitions observed in the near-IR would help define the structure of the iron environment more eas ily . However, one disadvantage of d-*d spectroscopy of Fe is the requirement for high-spin ferrous ion, where the spin-allowed excitation can occur in e ith er octahedral or 5 5 5 5 tetrahedral ligand fie ld ( T2g -FEg and Eg+T2g respectively). Near-IR spectroscopy has been reported for 1-Fe, 2-Fe, and 4-Fe p r o t e i n s . ^ '72,74,75 ^ case of 4-Fe reduced HIPIP, the near-IR absorption does not detect transitions analogous to 1-Fe or 2-Fe 6 p r o t e in .^ The results were interpreted to be caused by electronic del oca Iiz a tio n , making the four Fe atoms nearly equivalent and the assignment of Fe( I I ) or Fe( I I I ) to each Fe atom of the 4-Fe cluster impossible. The near-IR absorption spectroscopy is hindered by the intense interference of vibrational absorption of h^O at ^T.3vi, despite the substitution with D2 O which can a lle v ia te th is problem considerably. Uni ike absorption spectroscopy, vibrational CD is a weak phenomenon,^ and the near-IR CD spectrum only detects electronic excitations. This has helped in the theoretical interpretations of spectroscopic results. The near IR CD has supported the tetrahedral structure of 74 the Fe atom In rubredoxin, and the interactions of the bridged Fe 78 atoms in 2-Fe ferredoxin. 1.4. Magnetic Circular Dichroism Magnetic circ u la r dichroism or MCD is c irc u la r dichroism induced by a magnetic fie ld parallel to the light beam. Natural CD requires the presence of molecular c h ir a lity which is not present in the m ajority of molecules. In contrast, MCD has no such requirement, and exists in any molecule. The MCD phenomenon is caused by the Zeeman e ffe c t of a longitudinal magnetic fie ld to the electronic tran sition of a molecule. MCD is contributed by three phenomenon, called A, B, 79 and C terms. A term is the resu lt of the Zeeman s p littin g of either ground or excited states into le ft and rig h t c irc u la rly polarized components. B terms arises from the mixing of electronic states. C 7 term is caused by the Zeeman e ffe c t to a degenerate ground state and 79 the d iffe re n tia l population of these sublevels. A and B terms are 79 T-independent, while C term is proportional to 1/T. MCD is a signed quantity like CD and proportional to the intensity of the applied magnetic f ie ld . MCD are often measured with a magnetic fie ld ranging from 10 to 50 kilogauss. Such fie ld s are provided by either an electromagnet or a superconducting magnet. The MCD spectra are often reported as d iffe re n tia l molar c o e ffic ie n t per Tesla (kilogauss) or Ae/T. The near IR-visible-near UV MCD of the d iffe re n t iro n-sulfur proteins has led to the conclusion th at: a) MCD is a function of cluster structure and oxidation state, b) MCD is insensitive to protein environment. While conclusion a indicates that MCD is not a n a ly tic a lly sim ilar to CD, b shows the difference between the two methods. The MCD of d iffe re n t 4-Fe proteins are identical with 51 52 respect to th e ir oxidation states. * A major change of MCD 2- 3- 51 52 magnitude and form occurs on going from a C to a C state. 9 However, the results are less clear in the case of 2 Fe-2 S 51 52 clu ster. 9 The MCD of 2-Fe protein is at least one magnitude smaller than that observed in the 4-Fe cluster. 1.5. CD and MCD of N^ase Components The systematic CD and MCD studies of iron-sulfur proteins have shown to be of special value in characterizing cluster type, its oxidation state and protein e n v iro n m e n t.^ '^ Unlike EPR and 8 Mossbauer spectroscopy, CD/MCD studies do not require cryogenic sample freezing. They have the advantage of being measured in solution, and at pH and temperature relevant to the physiological function of the protei n. Since the FeMo and Fe components contain iron-sulfur clusters, it would be of interest to apply the CD/MCD techniques to the study of l^ase. One important aspect of the CD/MCD studies of l^ase is the demand for gram quantities of purified enzyme. With e ranging between 2 4 1 0 -1 0 in the near IR -visible-near UV region, optimal spectroscopic results requires a concentrated protein sample, as well as d iffe re n t cells of various light paths. In addition, highly active and purified enzyme preparations are desired because impure and less active 45 49 preparations could lead to a rtifa c tu a l resu lts. 9 One chapter is devoted to the multigram preparation of highly active and pur ifife d l^ase components from A. vinelandi i . The chapter which follows reports near IR -visible-near UV (3000-200 nm) CD/MCD spectra of the d ith io n ite - reduced l^ase components, as we I I as the diagnostic spectra for Fe-S cluster structure in reduced and oxidized Av2. The final chapters are devoted to the visib le -n e ar UV CD of the Av2 component. Chapter 4 presents the medium and temperature effects on reduced Av2. The medium perturbation includes the variations of ionic strength, pH, and buffer. The temperature was varied from 4°C to 40°C. Since inactivation of Av2 has been the source of misleading 45 49 results, ' chapter 5 describes the comparative studies between the bathophenanthroline and CD methods as monitors for C^-inactivated Av2. Chapter 6 reports a new property of Av2 called "se lf-o x id a tio n ” . This 9 process was found to accelerate in the presence of ATP or ADP. A series of experiments were performed to investigate the p o s s ib ilitie s of C^-leakage, d ith io n ite decomposition, and AvI contamination as causes for Av2 "self-o xid atio n ". Chapter 7 reports the use of CD to monitor the t it r a t i o n of oxidized Av2 with eith er ATP or ADP. The number of binding sites and equilibrium constants were determined from the t it r a t io n results. 10 Figure 1-1. Structures of 1-Fe, 2-Fe, 4-Fe, and 3-Fe Iron-Sulfur Clusters. A. 1 Fe-S cluster B. 2 Fe-S cluster C. 4 Fe-S cluster D. 3-■Fe cluster. / I ► F e 1 r SR i SR / RS- RS V \ S / V SR CHAPTER 2 MULT I GRAM PURIFICATION OF AZOTOBACTER VINELAND I I NITROGENASE PROTEINS 2 .1. Introduction The p u rificatio n of nitrogenase proteins from Azotobacter vinelandi i or from other procaryotic organisms has been a challenging process due to th e ir oxygen s e n s itiv ity . The c a ta ly tic a c tiv ity of the enzyme is rapidly and irre v e rs ib ly reduced by the presence of oxygen.^ Necessary anaerobic handling of nitrogenase has influenced many aspects of its isolatio n , namely the addition of extra steps to enhance pu rity, the scale, and the storage. Past e ffo rts to purify nitrogenase can be c lassifie d into two methods. Method I involves the co-purification of the nitrogenase components until the last step when 8 80 81 they are separated by DEAE-celIulose chromatography. ' ' This method exploits the r e la tiv e s t a b ilit y of the nitrogenase complex compared to its separated components. A. vinelandi i nitrogenase obtained by th is method^ has low a c tiv ity (Av1 SA 313, Av2 SA 1678) and is less pure. C ry s ta lliz a tio n of Av1 increases the SA to 1 4 5 0 .^ However, heme bands at 420, 525 and 557 nm are noticeable in the v is ib le absorption spectrum of th is c ry s ta llize d Av1 . ^ Method I was therefore abandoned in favor of Method I I . In Method I I , the 13 nitrogenase components are i n i t i a l l y separated and purified individually in subsequent steps. B r ill and Shah developed Method II and obtained c ry s ta llin e Av1 of SA 1638 and Av2 of SA 1 8 1 5 .^ However, the rep etitio n of these results has not been frequently attained, as evidenced by later publications. Lower specific a c tiv itie s were reported by B r ill and Shah in th e ir kinetics studies (AvI SA of 1125 and Av2 SA of 1 0 6 0 ) .^ Quoting the procedure of B r ill and Shah, Kleiner and Chen reported specific a c tiv itie s of 900-1100 in th e ir analytical studies in 1974.^ The d if f ic u lt y seems to be caused by the preparative electrophoresis step where anaerobic conditions cannot be achieved ea s ily . In addition, milligram yields of the nitrogenase components by method II are not practical to spectroscopic studies. The chiroptical studies of nitrogenase components demand a large quantity of protein (gram amounts). Optimal purity and specific a c tiv itie s are also essential, since low -activity and impure preparations could lead to d iffe re n t resu lts. This chapter describes a new nitrogenase p u rificatio n method (published in reference 83) which yields routinely gram amounts of A. vinelandi ? nitrogenase components of high purity and specific a c tiv ity . 2 .2 . Equipment - Aminco 4-338 rap ? d— f iI I 20,000 psi French pressure cell mounted on 5-598A Aminco Laboratory Press. - Beckman J-21B refrigerated centrifuge. 14 - Beckman rotors and centrifuge bottles models: JA21, JA20, JAM, and JA10. - Beckman Futura pH electrode, Model 39504 capable of measuring pH of Tris buffer. - Pharmacia glass columns, Models: K100/45, K50/100, and K50/30. - Pharmacia gradient mixer Model GM-1. - Pharmacia p e r is ta ltic pump Model P-3 for use with K50 glass columns. - Pharmacia UV-monitor capable of measuring absorption at 440 nm. - Pharmacia Servo-Graphic recorder Model 410. - Co I e-Parmer Masterflex pump equipped with Model 7014 pump head for use with tubing of ID" x 0D" = 0.0655 x 0.1945. - Am Icon u l t r a f i lt r a t i o n c e lls Model 402, 202, and 52. - Am icon u l t r a f i lt r a t i o n membranes: PM-30 and XM-50. - Engelhard Deoxo-Gas p u r ifie r , Model D for removal of O2 from H^ gas used to pressurize u l t r a f i It r a t i o n c e lls . - A glove-box custom-made by Van Beek Industries (New Jersey) and flushed with N2 scrubbed by Ridox (F is h e r). A special port f it te d with a gc-type stopper was installed to the side of the glove box to f a c i li t a t e anaerobic transfer of samples into or out of the box. - Radiometer Copenhagen conductivity meter and conductivity c e l l , model CDC 314. - Varian 1440 aerograph equipped with flame ionization detectors. - Varian 485 integrator. - Beckman Acta VI UV-v i s spectrophotometer. - Beckman Spectro 24 spectrophotometer. 15 2 .3 . Chemicals Trizma-HCI (264.4 gm) and Trizma-base (38.8 gm) were mixed in 2 I of deionized water to make a stock solution of 1 M T ris at pH 7 .4. The elution buffers: 0, 0 .1 , 0.15, 0.25, and 0.25 M NaCI in 0.025 M T ris , pH 7.4 were prepared by d ilu tin g 1 M Tris and adding dry NaCI. They were tit r a te d to pH 7.4 with eith er 1 N NaOH or 1 N HCI. The conductivity of each buffer solution was c a re fu lly measured before use to ensure the rig h t s a lt concentration. All buffers were degassed and sparged with Ar during chromatography. Argon gas was 0 2 “ Purified by a 3+ Q3 Cr /amalgamated Zn metal bubbler. D ith io n ite reagent was prepared in 0.08 M solution in an serum-stoppered erlenmeyer fla s k . The solution, containing 40 ml of H2 O and 0.8 ml of 1 N NaOH, was sparged with Ar and cooled to ice temperature for 5-10 min before adding 0.562 gm of sodium d ith io n ite . The d ith io n ite was dissolved by shaking the flask under flushing Ar. The reagent is usually injected to buffers at 1-2^ v/v to make them reduci ng. Microgranular DE52 ion exchanger (Whatman) was conditioned by successive washings with 0.5 N NaOH, 0.25 M Tris (pH 7 .4 ) , and 0.025 M Tris (pH 7 .4 ) . The conditioned resin was stored at 4°C, suspended in 0.025 M T ris , pH 7 .4 . Protamine sulfate reagent was prepared by dissolving 25.2 gm of Salmine Protamine Sulfate Grade II (Sigma) in 600 ml of warm H2 O. The mixture, titr a te d to pH 6.0 with 1 N NaOH, was diluted to 1.26 ml with deionized water. The yellow and turbid protamine su lfate solution, 16 caused by histone im p u ritie s , was cen trifug ed fo r 1 hr at 1 0 , 0 0 0 rpm. The yellow p e lle t was discarded, and the c o lo rless and clear supernatant, labelled PS-R, was saved and stored at 4°C. PS-R is usually warmed up to room temperature before use. 2 .4 . Cel I Breaking Azotobacter vinelandi i c e lls were grown in m ultikilogram amounts 84 85 by a continuous flow method. * The b a c te ria l c e lls were stored frozen in liq u id nitrogen or a t d ry -ic e tem perature. Two kilograms of c e ll paste were thawed fo r each p u r ific a tio n . Thawed c e lls were washed in an eq u ivalen t volume of 0.025 M T r is , pH 7 .4 . The homogenized m ixture was cen trifug ed at 10,000 rpm at 4°C fo r 10 min. The lig h t brown supernatant was discarded. The grey and orange top layers of the p e lle t were also removed. The tan p e lle t was resuspended in an equ ivalen t volume of cold (4°C) 0.025 M T r is , pH 7 .4 . The suspensate (^3 I in volume) was kept in an ice-w ater bath during c e ll breaking. Cell breaking was done at 16,000-20,000 psi with an Aminco French press equipped with a rapid flow c e l l . Ruptured c e lls appeared dark brown in co n trast to the tan color of in ta c t c e lls . The e x tra c t was co lle c te d on ice and cen trifug ed a t 10,000 rpm in a JA-10 Beckman rotor at 4°C for 8-10 hrs overnight to ensure good sedimentation of c e ll d e b ris . The p e lle ts were m u ltila y e re d , the supernatant, calle d crude e x tra c t or CE-S, was dark brown and tu rb id . The pH of CE-S was immediately adjusted from 6 . 6 to 7 .4 with 1 N NaOH. A small volume of CE-S (5 mis) was saved and stored frozen in 17 liq u id nitrogen for la te r a n a ly tic a l works (gel electro p h o resis, a c t iv it y assays, and b iu re t d eterm in atio n ). 2 .5 . Protamine S u lfa te P re c ip ita tio n The amount of protamine s u lfa te reagent or PS-R necessary fo r the p re c ip ita tio n of nucleic acids and non-nitrogenase proteins in CE-S was determined by a p ilo t t i t r a t i o n of PS-R to CE-S. T ris b u ffe r was added to keep the to ta l volume constant a t 4 .5 mis. CE-S was introduced in equal amounts (3 mis) to a number of c e n trifu g e tubes (JA21 tu b e s ). T ris was added next, and PS-R la s t, followed by good vortex-m ixing. A fte r 10 min incubation, the tubes were centrifuged fo r 10 min a t 21,000 rpm. Each tube presented a p e lle t , increasing in size with increasing amounts of PS-R added, w hile the supernatant g radu ally lo st its dark brown c o lo r. Acetylene a c t iv it y assays were done on the d iffe r e n t supernatants. The units of a c t iv it y were p lo tted against the volumes of PS-R added (F ig u re 2 - 1 ) . A "cut" ju s t p rio r to ^2^2 ac" * ‘ ' v ^ " *‘y d ro p -o ff, was selected from the p lo t. The to ta l volume of PS-R and T r is -b u ffe r to be added to the main batch of CE-S was obtained from the equations: vol (ml) of PS-R = vol (ml) of CE-S x cut (ml of PS-R) 3 vol (ml) of T ris = vol (ml) of CE-S x cut (ml of T ris ) 3 18 The to ta l volume of PS-R added varied from 20-40/C of the CE-S volume. The concentration of proteins in CE-S and the concentration of PS-R were probably responsible fo r th is varian ce. PS-R was added with s tir r in g to CE-S a t room tem perature. White p re c ip ita te appeared immediately upon addition of PS-R. The reactio n was allowed to proceed fo r 10 min before c e n trifu g in g the m ixture a t 1 0 , 0 0 0 rpm fo r 20 min. The p e lle t was m u ltila y e re d . The supernatant, c a lle d PS-S, remained dark-brown and tu rb id . PS-S was decanted into a sealed 4 I f i l t e r i n g fla s k and degassed under Ar for a t least three cycles. D ith io n ite was added to the deaerated PS-S at 2% v /v . The d ith io n ite-red u ced PS-S was stored in an ice-w ater bath and under Ar pressure before DEAE chromatography. PS-S was assayed fo r acetylene a c t i v it y . Full recovery of nitrogenase a c t iv it y was fre q u en tly obtained. All p u rific a tio n steps subsequent to the protamine s u lfa te p re c ip ita tio n step were performed under anaerobic con ditio ns. 2 .6 . F ir s t DEAE Column The nitrogenase components (FeMo and Fe) were fra c tio n a te d by a 10 x 30 cm column of DEAE-celIuIose. Two kilograms of m icrogranuIar DE52 (Whatman) were suspended in 0.025 M T r is , pH 7 .4 forming ^ 3 .5 I of th ic k s lu r r y . The m ixture was deaerated under Ar and made reducing by 3-4 m M of d ith io n ite . The presence of a c tiv e d ith io n ite in b u ffe r solutions was checked with redox dye in d icato rs (methyl viologen or methylene b lu e ). The d ith io n ite -re d u c e d DEAE c e llu lo s e was shaken and poured under a strong stream of Ar into a Pharmacia K100/45 glass 19 column. The column was immediately sealed and the resin was le t to s e t t le . Argon-sparged 0.025 M T r is , pH 7 .4 containing 1-2 m M d ith io n ite was pumped to the column to a c c ele rate the sedim entation. A Co I e-Parmer p e r is t a lt ic pump provided a packing flow ra te of 20-25 m l/m in. The eluted b u ffe r usu ally remained d ith io n ite reduced. A Pharmacia UV monitor equipped with two flow c e lls measured the absorption at 440 nm of chromatographed fra c tio n s (F ig u re 2 - 2 ) . PS-S was pumped under flu sh in g Ar at 20-25 ml/min to the packed column. I t took more than 2 hrs to load 3-4 I of PS-S. Next, the column was washed with ^ 2 I of 0.025 M T r is . A dark brown band remained bound to the upper h a lf of the column, w hile pinkish and tu rb id m a te ria ls were chased o u t. This pink fra c tio n was discarded. Following 0.025 M T r is , the column was successively washed with 0.10 M, 0.25 M, and 0.50 M NaCI in 0.025 M T r is , pH 7 .4 containing 1-2 m M of d ith io n it e . Brown and orange hemoproteins eluted out in 4 I of 0.10 M NaCI b u ffe r. They were saved and g ifte d to cytochrome researchers. FeMo p rotein was chased with 0.25 M NaCI b u ffe r as a dark brown band. I t was c o lle c te d in dearated vessels under flu shing Ar. Using the ^ 4 4 Q nm ^race f rom +he UV monitor (F ig u re 2 - 3 ) , the FeMo band was divided into th ree fra c tio n s : 200 ml o f precut c a lle d FeMolA, 700-1,000 mis of m ain-cut or FeMolB, and 700-1,000 mis of p ost-cut or FeMoIC. FeMolA and FeMolB were dark brown, almost black; FeMoIC was lig h t brown. The m ain-cut FeMolB contained most of the enzyme a c t i v it y , and w ill be used in the next p u r ific a tio n step. Traces of FeMo was washed out in 6 I of 0.25 M NaCI b u ffe r before sw itching to 0.50 M NaCI b u ffe r . Fe p rotein was removed from the 20 column in 0.50 M NaCI b u ffe r. The enzyme was c o llected in Ar-flushed vessels. Three cuts were chosen from the Fe band (Figure 2 -3 ): 400 mis of p re-cu t FelA, 400-500 mis of m ain-cut FelB, and 400ml of post-cut FelC. FelA was dark brown, FelB was greenish brown, and FeIC was lig h t brown. The FeMo and Fe fra c tio n s were s o lid ifie d into smaM p e lle ts by pumping them d ir e c tly into liq u id n itro g en . The p e lle ts were put into bags and stored in a liq u id nitrogen co n tain er. DEAE was cleaned with 0 .5 N NaOH. Purple and greyish proteins l e f t in the column were removed. The resin was reconditioned a e ro b ic a lly with an equal volumes of 0.25 M T r is , pH 7 .4 , followed by equal volume of 0.025 M T r is , pH 7 .4 u n til the pH s ta b iliz e d (1-2 washes). 2 .7 . Further P u rific a tio n of FeMo Component 2 .7 .1 . Heat Step and Second DEAE Column FeMoI was fu rth e r purifed by treatm ent a t 52°C followed by rapid cooling to ice tem perature. A system of two glass c o ils was set up for th is process to handle a large volume of FeMoI and keep the heating under anaerobic conditions (Fig ure 2 - 4 ) . The heating c o il was made of pyrex glass tubing of ID x OD of 5 x 6 .5 m m and composed of 20 s p ira ls of 20 cm diameter each. The cooling coil is sm aller and made of 9 turns of 10 cm diameter each. The heating co il was immersed in a 52° water bath, thermostated by a c irc u la tin g heater, w hile the cooling co il was buried in ice. The two c o ils were connected by a 21 p la s tic tu b in g . Enzyme and b u ffe r solutions were pumped through the c o ils to a valve which opened to the inside of a glove-box. Deaeration of the c o ils was done by washing them with d ith io n ite solutions and flushing them with Ar. FeMolB was thawed under Ar in a sealed f i l t e r i n g fla s k . M illim o la r d ith io n ite was injected to 700-1,000 mis of thawed FeMolB. The enzyme was pumped at W 6 ml/min through the c o ils by a CoI e-Parmer pump. This flow ra te allowed FeMolB to be heated fo r 5-6 min before being cooled and c o llected inside of the glove-box. The enzyme formed white p re c ip ita te when heated. Heated FeMolB was taken out of the glove-box in 0 -rin g sealed p la s tic b o ttle s fo r c e n trifu g a tio n . The b o ttle s were centrifuged by a Beckman J-21B c e n trifu g e a t 10,000 rpm fo r 20 min in a JA-10 ro to r, and introduced back inside of the glove-box. The dark-brown supernatant was c a re fu lly decanted into a 4 I beaker. The greyish p e lle t was discarded. The supernatant was d ilu te d 1:1 v /v with d ith io n ite -re d u c e d 0.025 M T r is , pH 7 .4 and c alle d HS-S. HS-S was pumped from the glove-box to a 10 x 30 cm column of d ith io n ite -re d u c e d DEAE. The resin was conditioned in 0.025 M T r is , pH 7 .4 . The to ta l volume of HS-S loaded on the column exceeded 2 I . Successive washes with 0.15 M NaCI and 0.25 M NaCI buffered a t pH 7.4 in 0.025 M T ris followed the loading. Both b u ffers contained 1-2 m M d ith io n ite and were sparged with Ar during e lu tio n . FeMo was chased out in 0.25 M NaCI b u ffe r. The enzyme was co llected in sealed b o ttle s under flushing Ar. The dark-brown FeMo band was divided in 3 fra c tio n s : 200 mis of brown p re-cu t c a lle d FeMollA, 'WOO ml dark brown 22 m ain-cut FeMollB, and ^700 ml of post-cut FeMoI 1C. FeMolIB contained most of the nitrogenase a c t iv it y . This fra c tio n was s o lid ifie d and stored frozen in liq u id nitrogen to be used fo r c r y s ta lliz a t io n . FeMol1A and FeMol1C can be combined with equ ivalen t fra c tio n s from another p u rific a tio n fo r c r y s t a lliz a t io n . Figure 2-5 is the e lu tio n p r o file of the second DEAE c e llu lo s e column. 2 .7 .2 . C r y s ta lliz a tio n of FeMo Component Most steps during the c r y s ta lliz a tio n of FeMo component were performed inside of a l^ -flu s h e d glove box. FeMollB was thawed under Ar and concentrated 5-6 fo ld s by u l t r a f i I t r a t i o n on PM-30 membrane. Two A m icon u l t r a f i l t r a t i o n c e lls Model 402 and 202, pressurized by were used to handle >700 ml of FeM olI. The NaCI concentration was decreased to 0 .0 4 -0 .0 2 M by d ilu tin g >100 ml of concentrated FeMolI with 500 ml of d ith io n ite-red u c e d 0.025 M T r is , pH 7 .4 . Next, the enzyme was concentrated 5-6x on XM-50 membrane. FeMo became muddy at elevated concentration, in d icatin g the presence of p r e c ip ita te . This fra c tio n was heated at 38° in a w ater-bath for 1 hour to increase c ry s ta ls form ation, in a sealed and Ar-flushed pyrex glass fla s k . Microscopic examination of the p re c ip ita te showed the presence of brown needle-shaped c ry s ta ls . FeMo was tra n s fe rre d back to the glove-box a fte r the 38° bath. The protein solution was put inside of O -ring sealed JA-20 c e n trifu g e tubes and taken out of the glove-box fo r c e n trifu g a tio n . FeMo was spun at 20,000 rpm fo r 20 min and returned to the glove-box. The dark-brown supernatant, c a lle d FeMo-S was saved for r e c r y s ta lliz a t io n . The dark-brown p e lle t was washed 23 with equal volume 0.025 M T ris and resuspended in ^100 mis of 0.25 M NaCI in d ith io n ite -re d u c e d 0.025 M T r is , pH 7 .4 . C e n trifu g a tio n at 2 0 , 0 0 0 rpm for 1 0 min followed each ad d itio n of solution to the p e lle t . The fin a l supernatant, c a lle d FeMoX, was stored as frozen p e lle ts in liq u id n itro g en . The wash and the greyish p e lle t were d i scarded. 2 .8 . Further P u rific a tio n of Fe Component 2 .8 .1 . Linear G radient Chromatography FelB was fu rth e r p u rifie d by a 0.25 M - 0.50 M NaCI lin e a r g radient DEAE column. DEAE was packed in d ith I onite-reduced 0.025 M T ris a t pH 7.4 inside a 5 x 20 cm Pharmacia glass column Model K50. FelB was thawed under Ar and d ilu te d 1:1 v /v with 0.025 M T ris containing 1-2 m M d ith io n ite . The d ilu tio n lowered the NaCI concentration to ^ 0 .2 5 M NaCI. The enzyme was subsequently loaded to the DEAE column a t 2 -2 .5 ml/min using a P-3 Pharmacia pump. The two rese rv o irs of a Pharmacia gradient mixer model GM-1 were f i l l e d (250 ml each) with d ith io n ite-re d u c e d 0.25 M NaCI and 0.50 M NaCI, both buffered a t pH 7 .4 by 0.025 M T r is . The apparatus was kept under N2 ~atmosphere, inside of the glove-box. The g rad ien t e lu tio n started immediately a fte r loading. Solutions were pumped from the glove-box to the column v ia a double-ended needle inserted through a septum sealed p o rt. The lin e a r change in NaCI concentration was measured by a Radiometer co n d u ctiv ity flow c e ll (in m S u n its) attached in lin e 24 with the flow c e ll of the UV-moni+or. Four colored p rotein bands were separated in the lin e a r s a lt g rad ien t column: grey, dark brown, blue and brown bands from top to bottom re s p e c tiv e ly . The dark-brown band situ ated in the 0 .3 -0 .3 5 M NaCI region contained the Fe component. The column was topped with 0.50 M NaCI b u ffe r to pump out a ll four protein fra c tio n s . The Fe fra c tio n was co llected inside of the glove-box and concentrated 5x on PM-30 with h^-pressurized A m icon U l t r a f i l t r a t i o n c e ll Model 202. The concentrated Fe fra c tio n , c a lle d F e l l , were stored frozen in liq u id n itro g en . The blue band, id e n tifie d as flavodoxin by absorption spectroscopy, was also c o llected and saved. The other two protein bands were discarded. The ^440nm " * Tace e lu tio n and the co n d u ctiv ity g rad ien t are presented in Figure 2 -6 . 2 .8 .2 . Sephadex G-100 Chromatography F e ll was chromatographed on a 5 x 80 cm column of Sephadex G-100 (Pharm acia). Superfine Sephadex G-100 was found to produce b e tte r sep aration, but reduced the flow ra te by a fa c to r of 5. The gel was packed under flushing Ar in 0.025 M T r is , 2 m M MgC^* 0.1 mg/ml DTT, and 2 m M d ith io n it e . F ell was thawed under Ar and loaded to the column with a Pharmacia P-3 pump. The protein was chromatographed in the same T ris b u ffe r a t 5 ml/min flow r a te . Sephadex G-100 separated F e ll into 3 colored pro tein s: grey, brown and dark brown from top to bottom re s p e c tiv e ly . The brown band contained Fe protein and was c o lle c te d inside of the glove-box. The p rotein was concentrated 5x by u I t r a f i I t r a t i o n on PM-30 membrane. This concentrated fra c tio n , c a lle d 25 FeG, was quickly injected in liq u id nitrogen to form frozen p e lle ts . The p e lle ts were stored in small bags in a liq u id nitrogen co n ta in er. The blue flavodoxin band was also co llected and saved. Figure 2-7 represents the e lu tio n tra ce of the Sephadex G-100 column. 2 .9 . P u rity Analyses 2 .9 .1 . Introduction Various a n a ly tic a l methods were used to determine the p u rity of the nitrogenase components a t each stage of the p u r ific a tio n . They are acetylene reduction assays, p ro tein assays, gel e le c tro p h o re s is, metal analyses, and MCD spectroscopy. Acetylene a c t iv it y was measured instead of Is^ a c t iv it y or a c t iv it y because i t is commonly used, thus perm ittin g d ire c t comparison with most published values. P rotein concentration was obtained by the b iu re t method. A n aly tical gel electropherosis showed the amount of im p u rities elim inated a t each step of the p u r ific a tio n . The number of Mo atoms and Fe atoms in FeMo and Fe atoms in Fe, determined by atomic absorption, is another c rite riu m for p u r ity . F in a lly , MCD spectroscopy was used as an a n a ly tic a l tool to detect traces of heme im p urities in the nitrogenase fra c tio n s . 26 2 .9 .2 . CqHq R eduction Assays 84 The d e ta ils of th is method were published. Except fo r the crude e x tra c t and the protamine s u lfa te PS-S fra c tio n , the acetylene a c t iv it y of each nitrogenase component was measured by c r o s s - t it r a t io n . Excessive amounts of FeMo, but not Fe, in h ib it nitrogenase a c t i v it y . The flow of electrons to the excess FeMo was believed to cause th is in h ib ito ry e f fe c t. ADP, a hydrolysis product of ATP during tu rn o ver, is also 86 in h ib ito ry to nitrogenase a c t iv it y . An ATP-generator composed of c re a tin e phosphate and c re a tin e phosphokinase was used to circumvent th is e f f e c t . ^ D ith io n ite is the electro n donor in the assay m ixtu re. The assay vessels are serum-stoppered to keep the reagent under Ar pressure. Each vessel contains 5 umoles of ATP, 25 limoles of c re a tin e phosphate, ^ 7 .8 units of c re a tin e phosphoki nase, 5 pmoles of MgC^* 25 ymoles of HEPES a t pH 7 .4 , 25 umoles of sodium d ith io n it e , and 1 ml of 84 acetylene. The in je c tio n of Fe which immediately followed th a t of FeMo, in itia te d the re a c tio n . The b o ttle s were incubated in 30° shaker bath, and injected with 0.25 ml of 25/6 w/v of tric h lo ro a c e tic acid to term inate the re a c tio n . A ty p ic a l protocol fo r acetylene assays of FeMo and Fe is shown in Table 2 -4 . The amounts of ethylene produced were measured by gas 84 chromatography, using acetylene as internal standard. Separation of ethylene from acetylene was obtained by a Porapak N column heated at 50°C. The GC data were calcu lated with a programmable HP65 or TI59 27 c a lc u la + o r. 2 .9 .3 . B iu re t Analysis The B iu re t method was found most r e lia b le fo r the determ ination of the protein concentration of isolated nitrogenase components. The b iu re t reagent was prepared from a procedure o u tlin ed in C.E. 81 McKenna*s d is s e rta tio n . The p rotein sample was dissolved in 0.9$ s a lin e to a to ta l volume of 1 ml. Four mis of B iu re t reagent were added next, followed by vortex-m ix in g . The OD readings at 555 m m were measured 30 min la te r . The b iu re t reagent was standardized with 5 mg/ml bovine serum albumin. The slope and in te rc e p t of the standard curve were used to c a lc u la te the p rotein con centration. 2 .9 .4 . Gel Electrophoresis The techniques and apparatus used in anaerobic gel electrophoresis fo r p u rity analysis of nitrogenase components were 8 8 85 published and described in d e ta ils elsewhere. Anaerobic conditions were necessary to prevent 0 2 “ damage of the nitrogenase 88 proteins which could give misleading e le c tro p h o re tic re s u lts . A Hoefer Model DE102 electro p h o resis u n it was modified to become hermetical (a p ic tu re of the modified apparatus is featured in references 8 5 ,8 8 ). The gel system, supported by a 5 x 75 m m IDXL glass tube, is composed of 1 cm of Iarge-pore gel topping 6 cm of sm alI-pore g e l. The gels were prepared according to the procedure 85 in itia te d by Davis. The large-pore gel holds 2.5$ acrylamide monomer and 0.625$ BIS c r o s s -Iin k e r , polymerized in 0.06 M Tris-HCI at 28 pH 6 .7 0 . The sm all-pore gel Is 7$ acrylamlde and 0.184/6 BIS, polymerized in 0.375 M Trls-HCI a t pH 8 .9 0 . The electro p h o resis u n it, with 8-10 gel tubes in place, was deaerated under Ar, f i l l e d with degassed 0.384 M g lycin e b u ffe r a t pH 7 .0 and 1 m M d ith io n it e , and * l e f t under flushing Av during the experim ent. Blue 85 guaiazule n e -3 -s u lfo n ate, serves as trackin g dye. Each protein sample contains 100-150 yg of p rotein dissolved in 40$ w/w sucrose, n,1 m M d ith io n it e , and 50 yM tra ckin g dye and was layered on top of the g e l . A DC cu rren t of 2 mA, generated by a Bio-Rad Model 400 power supply, is applied to each gel fo r ^2 hrs. The temperature was maintained below 20°C by c ir c u la tin g tap water through the cooling ja c k e t of the electropho res is u n it. The gels were removed from the glass tubes and stained in 1$ Ami do black (Bio-Rad) In 7$ a c e tic acid immediately a fte r elec tro p h o re s is . Destaining was done by a Bio-Rad Model 172 d iffu s io n destainer f i l l e d with 0-40$ methanol and 7$ a c e tic acid . The destained gels were stored in 7$ a c e tic acid for densitom etric analysis and photography. The FeMo and Fe proteins from each stage of the p u r ific a tio n were e le c tro p h o re tic a lIy analysed. The number of im purity bands decreases a t each successive p u r ific a tio n step. Im p urities are not v is ib le in the gel of FeMoX and FeG proteins (Fig ure 2 - 8 ) . Densitom etric an alysis showed th a t FeMoX is more than 99.9$ pure and FeG more than 99.0$ pure. 29 2 .9 .5 . Metal A n a ly s is The Mo and Fe contents have been fre q u en tly reported in prepara tive papers of nitrogenase^ as physical c h a ra c te riza tio n s of the p ro te in . The functional FeMo component is believed to contain 2 Mo and 22-38 Fe per molecule. The Fe component contains no Mo and 4 Fe per m olecule. Atomic absorption spectroscopy has the advantages of convenience and low detection lim it , and th e re fo re is more commonly used than the c o lo rim e tric methods fo r the determ ination of Mo and Fe in nitrogenase components. A Perk in-Elmer Model 306 Atomic Absorption spectrophotometer was used fo r the a n a ly s is . The furnace technique (flam e I ess atom ization) permits a detection lim it of 1 ppm of Mo and Fe. A liquots were injected by a 25 yI Eppendorf m ic ro -p ip e tte equipped with disposable t ip into a g rap h ite p y ro ly tic tube (P e rk ln -E Im e r). They were dried a t 125° fo r 30 sec, ashed a t 1400° fo r 30 sec, and atomized at 2800° fo r 15 sec. Mo was detected at 313.3 nm with a Molybdenum hollow cathode lamp (P e rk in -E lm e r). Fe was detected a t 248.3 nm with a hollow cathode iron lamp P e rk in -E lm er. Least-square f it t e d curves were obtained with 1000 ppm standardized solution of (NH^)gMo^0 2 ^ in H^O, and 1 0 0 0 0 ppm of standardized solution of Fe in HCI. Both solution s were from Anderson L ab o rato ries, Inc. (Texas). Protein samples were acid tre ate d and d ilu te d a e ro b ic a lly . Samples were made in d u p licate or t r i p l i c a t e amounts. The numbers of Mo and Fe atoms in FeMo and Fe p roteins are given in Table 2 -5 . They were con sisten t with other published re s u Its .^ 30 2 .9 .6 . D e te c tio n o f Heme Im p u ritie s by M C D Spectroscopy The CD and MCD of nitrogenase p roteins (discussed in the next chapters) were found to be s e n s itiv e to pther m e tallo p ro tein contaminants. In p a r tic u la r , the MCD in the v is ib le and near UV wavelengths can detect tra c e amounts of hemo p ro te in s , since th e ir M CD are much Iarger in magnitude and q u ite d is tin c t in form than iro n -s u lfu r proteins lik e the nitrogenase components. M CD spectroscopy can measure heme im p u rities in nitrogenase proteins at level v i r t u a ll y undetectable by ordinary absorption spectroscopy or gel electro p h o re s is . Av1 isolated by a m o d ificatio n of method I 82 orginated by Bulen and LeComte and adopted by th is Iaboratory p rio r to 1977, was found to e x h ib it considerable heme Soret (420 m m ) and Q (560 mm) bands (F ig ure 2 - 8 ) . Assuming the MCD of heme im p u rities to 89 be equal to th a t of ferrocytochrome C, the Av1 sample studied must contain ^ \% mole percentage of heme contaminant. Contamination at th is level is nearly unobservable by absorption spectroscopy. The M CD of Av1 had caused a reevalu a tio n of our p u r ific a tio n methods. The p u r ific a tio n procedure described in th is chapter, developed from the o rig in a l procedure of B r i ll and Shah, has proven successful in e lim in a tin g the traces of heme im p u rities in Av1. Heme bands were found to be non-existent^in the MCD o f c r y s ta llin e FeMoX (F ig u re 2 - 9 ) . 31 2 .1 0 . Cone I us i on The development of an A. vIneland i i nitrogenase p u r ific a tio n procedure described in th is chapter has established the follow ing important re s u lts : 1. C ry s ta llin e Av1 and homogeneous Av2 can be prepared- in multigram amounts with s ta te -o f-th e a r t s p e c ific a c t iv it ie s and p u rity . 2. The large scale p u r ific a tio n permits convenient and tim e-saving studies on a sin g le batch of enzymes, as we I I as other studies which req u ire considerable amounts of the nitrogenase proteins such as c h iro p tic a l stu d ies. 3. Both Av1 and Av2 contain no detectable heme MCD. 4. The proteins are ^99.9^ pure by a n a ly tic a l disc gel electrophores i s . 5. Metal analyses were con sisten t with other published re s u lts . 6 . This p u r ific a tio n procedure has been repeated fo r more than 10 times with reproducibly high SA and y ie ld s (see Table 2 - 5 ) . 7 . The procedure only requires two operators working fu ll tim e fo r no more than two weeks, excluding such time-consuming a n a ly tic a l te s ts such as gel e le c tro p h o re s is , metal an a ly s is , and MCD spectroscopy. 8 . The p o s s ib ility to store proteins w ithout loss of a c t iv it y between consecutive p u r ific a tio n steps has been of upmost advantage since most steps lasted 8-14 hours. 9 . The anaerobic gel electro p h o resis apparatus, and the d ith io n ite -r e s is ta n t trackin g dye developed in conjunction with 32 the nitrogenase p u r ific a tio n has been b e n e fic ia l to other workers with a ir -s e n s itiv e compounds. 10. A dry-box purged with gas was found most e ffe c tiv e for anaerobic handling of nitrogenase. 33 TABLE 2 -1 . PURIFICATION O F AVOP NITROGENASE. Whole C e lls (Aerobic) - Resuspend (0.025 M T r is , 4°) - Cen+rifuge (10,000 rpm, 10 min, 4°) P (Aerobic)- (Discarded) PS-P (Di scarded) (Di scarded) - Resuspend (0.025 M T ris ) - Break (French Pressure C e ll, 20,000 psi) - Cen+rifuge (10,000 rpm, 8 hrs, 4°) S (Aerobic) - Adjust pH to 7 .4 - Add protamine sulfa+e - Cen+rifuge (10,000 rpm, 20 min) PS-S - Degas, add 2 m M di+hion i+e - Load on DEAE-celIuIose column (10x30 cm) - Elu+e wi+h 0.025 M T ris -------------------------------Pink fra c tio n (c e ll debris) - Elu+e wi+h 0.10 M NaCI in 0.025 M T ris - Elu+e wi+h 0.25 M NaCI in 0.25 M T ris "Cytochrome” fra c tio n s Fe-Mo I fra c tio n SA = 300-500 - Elu+e wi+h 0.50 M NaCI i n 0 .25 M Tr i s Fe I fra c tio n SA = 700-800 34 TABLE 2 -2 . PURIFICATION O F FEMO COMPONENT. Fe-Mo I SA = 300-500 - Heat step: 5 2 °, 5 min - C entrifu ge (10,000 rpm, 30 min) HS-S HS-P (Di scarded) - D ilu te 2 -fo ld with 0.025 M T ris - Load on DEAE-celIuIose column - E lute with 0.025 M T ris - E lute with 0.15 M NaCI in 0.025 M T ris - E lu te with 0.25 M NaCI in 0.025 M T ris P re-cu t no. 1 P re-cu t no. 2 Fe-Mo I I fra c tio n SA = 600-800 - Concentrate 5 -fo ld (PM-30 membrane) - D ilu te 6 - f o ld with 0.025 M T ris - Concentrate 6 - f o ld (XM-50 membrane) - Heat step: 3 8 °, 1 hr - C en trifu g e (10,000 rpm, 10 min, room temp.) ----------------— S (Fe-Mo S) SA = 300-400 - Wash with 0.025 M T ris - Saved fo r r e c r y s ta lliz a tio n - C entrifu ge (20,000 rpm, 10 min, room temp.) S (Fe-Mo S’ ) - Combine u n it FeMoS - Dissolve with 0.25 M NaCI - C entrifu ge (20,000 rpm, 30 min, room tem p eratu re). (Discarded) ■ S (Fe-Mo X) SA = 1,800-2,000 (c ry s ta l Iin e ) 35 TABLE 2 -3 . PURI F€ SA = 1 FI CAT ION OF FE COMPONENT. » I r 00-800 - D ilu te 1 -fo ld w ith 0.025 M T ris - Load on DEAE-celIulose g radient column: 0.25 M NaCI 0 .5 M NaCI in 0.025 M T r is . (1) Greyish-brown (2) Fe fra c tio n SA = 1 II fra c tio n (3) Blue F^lavodoxin ,200-1,500 - Concentrate 5 -fo ld (PM-30 membrane) - Load on G-100 Sephadex column - E lu te with 0.025 M T r is /0 .0 0 2 M M gC^/O -l mg/ml d it h io t h r e it o l/2 m M d ith io n ite i ... (1) Grey fra c tio n (2) D il F (SA=1,5 Note: A ll steps are anaerobic i A ll buffers have a pH of S = supernatant, P = pel I A ll s p e c ific a c t iv it ie s duced per min per m g of protei ----------- . ( uted Fe-G fra c tio n (3) Blue Flavodoxin (Brown) - Concentrate 5 -fo ld (PM-30 membrane) - Load on G-100 column eG 0 0 - 2 , 0 0 0 ) in less otherwise in d icated . 7 .4 . e t . (abbreviated SA, in nmoles of pro- n) are obtained a t 95^ C2 H2 s a tu ra tio n . 36 TABLE 2 -4 . PROTOCOL FOR ACETYLENE ASSAYS. FeMo Assays Fe Assays B o ttle No. 1 2 3 4 5. 1* 2* 3* 4» 5» H2 0 , ml ATP-30, ml ■ < -------------- 0.40---------------- ^ --------------- 0 .4 0 --------------------► ■ Pump/Argonate X3 Vent DT-20, ml - < -------------- 0.25----------------- *.-----«------------------ 0.25 ------------------- Vent C 2 H 2 , m l------------------■ <------------- 1 .00 ------------ — > <---------------- 1 .00 ---------------- Vent FeMo, y la------------------ < --------------------- 2 — -------------c 5 > Fe, yla 5 10 20 40 100 10 20 30 40 50 Atime, min* 5 1 0 ----------------- ^ 1 0 -------------------- TCA, ml--------------------- ^--------------- 0.25 ■ < ----------------- 0.25 nmoles of C0 H. 2 4 units of mg/ml N2ase SAd volume a r b i t r a r i l y assigned. k reactio n tim e. c nmoles of produced per min. ^ un its per m g of p ro te in . 37 TABLE PROTEINS 2 -5 . PURIFICATION OF FE-MO AND FE FROM 2 KG OF AZ0T0BACTER VINELAND I I . Step Fraction V o l. Total A c tiv ity Total Protein SAa P u rifica tio n Yield (ml) (units) (mg) (units/mg) factor (%) Fe-■M o Protein 1 . Crude Extract (CE-S) 3,460 6.69x106 1.52x105 44 1 1 0 0 2 . Protamine Sulfate Fractionation (PS-S) 5,480 6.71x106 1 .37x105 49 1 1 0 0 3. DEAE-Cel1u lo se-1 (Fe-Mo 1) 1 , 0 0 0 1.27x107 2.86x104 444 10 1 0 0 c 4. 52°C Supernatant (HS-S) 1,250 1 .19x107 1.63x104 730 17 93c 5. DEAE-Cel1u lo se-11 (Fe-Mo II) 800 1.07x107 7.43x103 1 ,050 24 84c 6 . Crystal 1ized (Fe-Mo X) 2 0 0 7.47x106 5.44x103 2,000 45 60 O J 00 TABLE 2 -5 . (CONTINUED) Step Fraction Vo I . Total A c tiv ity Total Protein SAa Puri f icat ion Yield Fe Protein (m l) (units) (mg) (units/mg) factor (%) 1 . Crude Extract (CE-S) 3,460 6.69x106 1.52x1 O'’ 44 1 1 0 0 2 . Protamine Sulfate Fractionation (PS-S) 5,480 6.71x106 1 .37x105 49 1 10 0 3. DEAE-Ce11u1o s e -1 (F e - 1) 400 6.04x106 8.50x104 710 16 94 4. DEAE-Cel1u lo se-1 1 ( F e - I 1) 2 0 0 5.34x106 3.79x103 1,410 32 80 5. G-100 Sephadex (Fe-G) 10 0 4.38x106 2.30x103 2 , 1 0 0 48 72 aSA = ^Units specific a c tiv ity (nmoles of ethy = nmoles of ethylene formed/min. lene formed/m in per m g of protein) • c FeMo protein saturated with Fe protein • 0 4 VO TABLE 2 -6 . M O AND FE COMPOSITIONS OF AV1 AND AV2 PROTEINS. P rotein FeMo-X FeMo-Xa Fe-G Fe-G 9 SAU 1900-2000 1700-1800 1800-2000 1600-1800 Mo/mo I ecu Ie * 2 .0 + 0 .5 1 . 8 + 0 .5 Fe/moI ecu Ie ( 33.1 + 2 . 2 31 .5 + 0.17 4 .7 + 1.3 3 .7 + 0.54 a. proteins from another p u r ific a tio n . b. s p e c ific a c t iv it ie s obtained a t less than 95$ ^ ^ 2 sa’* ‘ura'f"'on • c. M W used were 240,000 for Av1 and 64,000 fo r Av2. d. Analysis performed by C.E. Kampe. 40 Figure 2 -1 . P lo t of Units of A c tiv ity as a Function of added PS-R. Assay vessel contains 5 ymoles of ATP, 25 ymoles of c re a tin e phosphate, 7 .8 units of phosphokinase, 5 yM HEPES a t pH 7 .4 , 25 yM of DT, and 1 ml of C ^ . The in je c tio n of 100 I (40 mg/ml) of PS-S in it ia t e s the re a c tio n . Units of a c t iv it y are expressed as nmol of C2 H2 produced per min. 41 Units o f Activity cut 120 O o 80 40 O z z Q . 0.4 1 .2 2.0 Volume of P S - R Added (mis) Figure 2 -2 . Experimental Set-Up of the F ir s t DEAE-CelIulose Column Chromatography. A. Ar-sparged e lu tio n b u ffe r. B. Pump. C. Pharmacia K 100/50 column. D. UV monitor (R = reference c e ll; S = sample c e l l ) . E. C o lle c tio n fla s k , flushed with Ar. Ar E Figure 2 -3 . E lu tio n P r o f ile of the F ir s t DEAE-CelIulose Column. Av1 and Av2 fra c tio n s were separated on DEAE-celIulose packed inside of a Pharmacia K100 column. The proteins were monitored by th e ir absorbance a t 440 nm by a Pharmacia UV-monitor. Each component was c o lle c te d into p re -c u t, m ain-cut, and p o st-cu t according to the e lu tio n peak. Elution time (hrs) 1 2 3 4 FeM o Protein 8 6 Pink fraction m i nl-cut 440nm 4 Fe Protein Hemo-proteins 2 pre-cur. post-cut pre-cu iost-cut 0 Figure 2 -4 . Experimental Set-Up of the Heat Step on FeMo Component. A. A r-flushed enzyme fla s k . B. Pump. C. Heating glass c o il immersed in 52°C water bath. D. Cooling c o il immersed in 0°C ice-w ater bath. E. Valve which opens to the inside of a glove-box. F. Vent v a lv e . 47 Q J > o | X o t-CS s_ <c 48 Figure 2 -5 . E lution P r o f ile of the Second DEAE-CelIulose Co Iumn. Av1 fra c tio n s were p u rifie d fu r th e r, follow ing the heat step treatm ent on DEAE-ceI Iulose packed inside of a Pharmacia K100 column. Av1 e lu tio n was monitored by the absorbance at 440 nm, measured with a Pharmacia UV m onitor. The protein c a lle d FeM oll, was c o lle c te d into three separate fra c tio n s lab elle d FeMolIA fo r p re -c u t, FeMollB for m ain-cut, and FeMoC fo r p o s t-c u t. 49 Elution time (hrs) .8 F e M o Protei n 0.6 440nm m ai nvcut 0.4 0.2 post-cut pre-cut Figure 2 -6 . E lu tio n P r o f ile of the Linear G radient Column on Fe Component. Av2 fra c tio n s were chromatographed on a lin e a r g rad ien t from 0 .2 5 -0 .5 M NaCI column, packed with DEAE-celIulose. The g rad ien t was generated by a GM-1 Pharmacia grad ien t m ixer, and measured in m S u n its by an o n -lin e c o n d u ctiv ity c e ll (Radiometer Copenhagen). The e lu tio n was monitored by the protein absorbance a t 440 nm. Fe p rotein was co lle c te d into 3 fra c tio n s c a lle d : p re-cu t (A ), m ain-cut (B ), and p o st-cu t ( C ) . 51 Elution time (hrs) Flavodoxin Protein Grey Protein Linear Gradient VJ1 N ) Figure 2 -7 . E lu tio n P r o f ile of the Sephadex G-100 Column. Lin ear-G rad ien t chromatographed Av2 was p u rifie d on Sephadex G-100 packed inside of a Pharmacia K50/100 column. The e lu tio n was monitored by the p rotein absorbance a t 440 nm. Fe protein was c o lle c te d in th ree fra c tio n s : p re -c u t, m ain-cut, and p o s t-c u t. 53 Elution time (hrs) Fe Protein A main-cut 440nm Grey Protein pre-cu post-cut FIavodoxin Figure 2 -8 . Anaerobic D isc-E Iectro p h o resis P attern s of P u rifie d FeMo and Fe F ra c tio n s . L e ft: FeMo p ro tein (40 yg of FeMoX). R ight: Fe pro tein (30 yg of FeG). The samples were run an a ero b ically fo r 2 hours a t 2 m A per tube. 55 56 Figure 2 -9 . V is ib le MCD Spectrum of S em i-P urified Av1. The Av1 solution contains approxim ately 5 mg p ro te in /m l, dissolved in 0.25 NaCI, 0.025 M T r is , pH 7 .4 , and 1.6 m M in d ith io n it e . The c e ll pathlength is 1 mm. The spectrum is shown a t +40 kilogauss (k g ). Av1 has SA less than 1600. 57 ( uju)x O O Q OOZ 009 0 1 = v v 31AI3H t? ~ 3IAI3H Figure 2 -1 0 . V is ib le MCD Spectrum of C r y s ta llin e Av1 . The Av1 so lu tio n contains 22.0 m g p ro te in /m l, dissolved in 0.25 M NaCI, 0.025 M T r is , pH 7 .4 , and 1.6 m M in d ith io n it e . The c e ll path length is 1 mm. The spectrum is shown a t ±40 kilogauss (k g ). Av1 has SA more than 2000. 59 .r 4 AA H = - 40.0 k g 600 700 400 500 X (nm) o* o CHAPTER 3 CD AND MCD OF NITROGENASE PROTEINS 3 .1 . Introduction The arrangements of the metal chromophores in nitrogenase proteins and th e ir c a t a ly t ic ro le remained to be elu cid ated despite extensive studies by several techniques such as EPR, Mossbauer, 39-50 51 52 EXAFS, and c lu s te r ex tru s io n . ' The c h iro p tic a l studies of iro n -s u lfu r proteins have been shown to be useful in character Izing c lu s te r typ e, oxid atio n le v e l, and p ro tein environment of these 71 77 p ro te in s . ' The CD (c ir c u la r dichroism) and MCD (magnetic c ir c u la r dichroism) of the ferredo xin s are more structured and s e n s itiv e to th e ir o xidation states than th e ir fe a tu re le s s absorption sp e ctra . CD 90 of nitrogenase has been obtained in the p rotein region (^300 nm) . CD d etection of the nitrogenase components a t longer wavelengths has not previously been achieved. The CD spectra in the v is ib le range of nitrogenase components from Azotobacter chroccocum and K le b s ie lI a 90 91 pneumon? a were reported v i r t u a ll y in e x is te n t. * The MCD of nitrogenase proteins have not been studied p rio r to th is work. The published CD and MCD studies of d Ith io n ite -re d u c e d FeMo and Fe, and FeMo-oxidized Fe across the near in fra re d -v is ib le -n e a r u lt r a v io le t 92 spectral regions are w ill be described in th is chapter. 61 3 .2 . Chemicals - Argon, Ni+rogen, and Hydrogen ( a ll from A irco , p re p u rifie d grade) - HEPES (N -2-hydroxyethyI p ip e ra zin e -N *-e th a n e s u Ifo n Ic a c id , C alb io - chem, UI fro I grade) - D i+hionl+e reagent was prepared as described in 2 .3 and added at 2-3/6 v /v to b u ffers and p ro te in s . - NaOH, NaCI, and MgCI 2 * 6 ^ 0 ( a ll from Mai Iin c k ro d t, AR grade) - D ith io th r e ito l (abbreviated DTT, from Sigma or Bio-Rad) - D2 O (99.7$ isotopic pure, from Merck, Sharp and Dohne) - DI sodium Adenosine Triphosphate d ihydrate (abbreviated ATP, from Sigma, Grade I I , 95-98/6 pure, c r y s ta llin e ) - Disodium C reatine phosphate te tra h y d ra te (abbreviated CP, from Pierce) - C reatine Phosphokinase (abbreviated CPK, Sigma, from ra b b it muscle, type I, M 55 units/m g ). 3 .3 . Reagents P rotein samples were buffered a t pH 7 .4 w ith 0.025 M Hepes. Dry 93 NaCI was added to b u ffe r a t 0.25 M fo r Av1 and 0.50 M for Kp1 93 samples. 0.1 mg/ml DTT and 2 m M MgC^ were added to Av2 and Kp2 samp Ie s . Samples for IR spectroscopy were prepared in D 2 O. Dry HEPES was dissolved in D2 O and t it r a t e d to pH 7 .8 with 1 N NaOD. The pipets and syringes used were rinsed with D2 O to avoid contam ination with H2 O. 62 93 D2 O b u ffe r for Av1 and Kp2 contained 0 .50 M NaCI. D2 O b u ffe r for Av2 and Kp2 contained 0.1 mg/ml DTT and 2 m M M gC^. D ith io n ite -re d u c e d proteins contained 1-2 m M of d ith io n it e . The reagents fo r enzymic o xid atio n of Av2 were made in 0.025 M HEPES, 0.1 mg/ml DTT, and 2 m M MgC^ and t it r a t e d to pH 7 .4 with 1 N NaOH. Both H2 O and D2 O samples of oxidized Av2 contained 20 m M of c re a tin e phosphate, 10 m M ATP, 1 mg/mi o f c re a tin e phosphokinase, and . 94 1% mole:mole of FeMo. The reagents were deaerated under Ar and made reducing with M . 6 m M d ith io n it e . 3 .4 . Equipment The proteins were handled inside of a glove-box from Van Beek In d u s trie s , equipped with a septum-sealed port which perm its the tra n s fe r of solutions in and out of the glove-box. The box was continuously purged with R id o x -p u rifie d n itro g e n . The absorption over the 200-2000 nm range was measured with Cary 17 spectrom eter. Cary 61 spectrop olarim eter measured the CD and MCD over the 200-800 nm range. CD and MCD in the near IR (700-2000 nm) were measured with an instrument constructed a t the U n iv e rs ity of 95 Southern C a lifo r n ia . Magnetic fie ld s up to 50 kilogauss (5 Tesla) were provided by a Varian superconducting magnet. The exchange of b u ffe r so lu tio n in p ro tein sample was done over PM-30 membrane in an Amicon 8 MC d i a f i I t r a t i o n u n it, pressurized with H2 which was scrubbed by a d e o xo p u rifier c a rtrid g e from Englehard Industr i e s . 63 C y lin d ric a l quartz c e lls (O ptical Cell Co.) of 11 m m internal diameter and pathlengths ranging from 0 .5 to 5 m m with in fr a s il or suprasil fused quartz windows were used fo r spectroscopic measurements. The c e lls were f i l l e d inside of the glove-box and enclosed in O -ring sealed c y lin d ric a l holders, equipped with c ir c u la r fused quartz windows (Heraeus-Am ersiI) . The c e lls were brought out of the glove-box inside of g a s -tig h t holders fo r spectroscopic measurements. A diagram of the c e ll and c e ll holder is shown in Figure 3 -1 . The absorption spectra are reported as e (molar e x tin c tio n c o e ffic e n t), the CD and MCD spectra are shown as d iff e r e n t ia l molar e x tin c tio n c o e ffic ie n t Ac and Ac/T (per Tesla) re s p e c tiv e ly . The M W of the proteins used in c a lc u la tio n s are 240,000 fo r Av1 , 64,000 fo r Av2, 218,000 fo r Kp1, and 67,000 fo r Kp2. 3 .5 . P rotein Sample Preparations The proteins were thawed from frozen p e lle ts under Ar and handled inside of the glove-box. Av1 of SA 2000 and Av2 of SA 1900-2000 were p u rifie d as described in Chapter 2 . Kp1 and Kp2 of SA 1700 and 1300 re s p e c tiv e ly were provided by B.E. Smith from the A rg ic u ltu ra l Research U n it, U n iv e rs ity of Sussex, Brighton, U.K. Concentration of the p ro tein s and solvent exchange were done by d i a f i I t r a t i o n over PM-30 membrane in an A m icon 8 MC u n it pressurized by O rig in a lly in T ris b u ffe r , the proteins were d ia f ilt e r e d into 0.025 M HEPES b u ffe r . Adjustments of the protein concentration and 64 app ro priate c e ll pathlengths were necessary to optim ize signal to noise r a t io . Acceptable samples should have OD 0 .5 -2 .0 across the v is ib le -n e a r UV range. IR samples contained 50-80 mg/ml of p ro te in , 20-40 mg/ml of pro tein were adequate fo r experiments in the v is ib le -n e a r UV range. The absorption, CD, and MCD were monitored r e p e t itiv e ly a t 1-5 hr in te rv a ls ( a t least tw ice) to ensure tim e-independent sp ectra. The measurements were disregarded in case of s ig n ific a n t changes of the spectra over the tim e of study. Acetylene reduction assays were measured p rio r and subsequent to spectroscopy studies to ensure f u ll re te n tio n of a c t iv it y over the tim e of the experim ent. The spectra of p roteins having less than 80/6 recovery of th e ir a c t iv it ie s were discarded. Av1 was c r o s s -titr a te d with Av2 and v ic e -v e rs a ; Kp1 was assayed with Kp2 and v ic e -v e rs a . The protein concentration was determined by the b iu re t or Lowry method. The Lowry’ s values were often 10-20 % lower than the b iu r e t ’ s values, p a r tic u la r ly in the case of FeMo p ro te in s . 3 .6 . Results and Discussion 3 .6 .1 . FeMo P roteins The absorption, CD, and MCD of d ith io n ite -re d u c e d (re fe rre d th e r e a fte r as reduced) Av1 and Kp1 are shown in Figure 3 -2 . CD and MCD of reduced Av1 and Kpl are observable across the near IR -v is ib le -n e a r UV range. Previous rep o rts on the non-existence of CD 65 in the v is ib le wavelengths of Ac1 and Kp1 has th e re fo re been contradicted by th is work. Weak observable CD of Kp1 in the v is ib le 90 range previously rep o rted , seemed ra th e r caused by the lack of instrumental s e n s it iv it y than the non-existence of c h i r a l it y in FeMo p ro te in . The Cary 61 spectropolarim eter is capable of measuring A -5 less than 10 . In a d d itio n , since the absorption decreases ra p id ly with increasing wavelengths from 2 0 0 nm, p rotein samples should reach 20-40 mg/ml for the v is ib le -n e a r UV studies and 50-80 mg/ml fo r the near-1R s tu d ie s . Also, various path lengths were necessary fo r optimal r e s u lts . F a ilu re to observe Kp1 CD in the v is ib le range by Eady e t 90 a l . was possibly due to in s u ffic ie n t absorbing sample since the protein concentration was much lower. The CD and MCD of Av1 and Kp1 in the 200-2000 nm range are v i r t u a ll y id e n tic a l. The resemblance in CD in d icates th a t the same protein -m o iety around the metal c lu s te rs e x is ts in the two proteins from d iff e r e n t n itro g e n -fix in g b a c te ria . In c o n tra s t to CD which r e f le c ts the c h ira l environment of the chromophore, MCD is s o le ly a property of the metal c lu s te r . Hence, the s im ila r it ie s between the two MCD of reduced Av1 and Kp1 imply th a t the metal c lu s te rs are s tr u c tu r a lly a lik e . The CD and MCD re s u lts are co n sisten t with the s im ila r it ie s in amino acid composition, and m etal/S ” atoms content of 96 the two p ro te in s . Besides, i t was found th a t heterologous enzyme m ixture such as Av1-Kp2 or Kp1-Av2 can give normal nitrogenase .. .. 26,96,97 a c t i v i t y . * 9 66 47 52 The recent Mossbauer and c lu s te r extrusion studies of FeMo pro tein suggested the presence of two types of metal c lu s te rs : M 4 c lu s te r containing Fe and Mo atoms, 6 and P c lu s te r containing Fe 48 46 atoms. M c lu s te r was believed to be a 4Fe-S c lu s te r . The n ear-IR MCD of reduced Av1 and Kp1, dim inishing to zero a t 1,000 nm, seemingly excludes the presence of a normal 4Fe-S c lu s te r , since i t usually e x h ib its s ig n ific a n t MCD in the 1 ,000-2,000 nm r a n g e . T h e CD of the two p ro te in s , several times sm aller in magnitude than the CD of 2-Fe fe rre d o x in s , ru le out the presence of such c lu s te r . In view of the large metal content in FeMo p ro tein (2 Mo, 24-32 Fe)^ i t is not possible to deconvolute the CD and MCD in to c o n trib u tio n s from d iff e r e n t c lu s te r types. The CD and MCD studies of FeMoco and the apoprotein would be useful -to th is purpose. 3 .6 .2 . Fe P roteins The absorption, CD, and MCD o f reduced Av2 and Kp2 are shown in Figure 3 -3 . They are the f i r s t spectra of the Fe component a t wavelengths longer than 760 nm. The reduced Fe proteins have featu re le s s absorption spectra, m onotonicalIy decreasing from the near u lt r a v io le t region to the near IR. This is also seen in most reduced 4-Fe and 8 -Fe ferre d o xin s . The CD of reduced Av2 and Kp2 are observable across the near IR -v is ib le -n e a r UV range. As with the FeMo component, these re s u lts have con tradicted previous reports on the 90 91 non-existence of CD in Fe component (namely Kp2 and Ac2) ' in the v is ib le range. The CD of reduced Fe p ro tein s are s im ila r in magnitude, but not in form, with the CD of 4Fe-S c lu s te r in 67 fe rre d o x in s . I t has been shown th a t the CD o f 4Fe-S c lu s te r v aries with p r o t e i n a n d th e re fo re is usually not d iagnostic of th is c lu s te r typ e. The CD of reduced Av2 and Kp2 are n early id e n tic a l. The resemblance in CD im plies th a t the p rotein environment of the metal c lu s te r is conserved in the two p ro te in s . The MCD of reduced Av2 and Kp2 are almost superimposable, suggesting th a t the two proteins contain s tr u c tu r a lly id e n tic a l c lu s te rs . Furtherm ore, the MCD is analogous in form and magnitude with the MCD o f the EFe4 $4 ( SFO4 IP or C3 c lu s te r in ferredox i ns The re s u lts were con sisten t with fin d in g s from c lu s te r e x tra c tio n , metal a n a ly s is , and Mossbauer stu d ies. Also shown in Figure 3 -3 , are the absorption, CD, and MCD of Av1-oxidized Av2, in the presence of an ATP g en erato r. These conditions perm it the rapid exhaustion of d ith io n ite and the o xidation 94 of Fe component as during enzyme tu rn o ver. The development of a ”390 nm” band in the absorption is in accord with other spectra of 2- oxidized 4-Fe C c lu s te rs . The CD has changed remarkabIy in form and magnitude. The d iff e r e n t ia l molar e x tin c tio n c o e ffic ie n t has almost quadrupled. Previous studies of 4-Fe c lu s te rs presented s im ila r re s u lts upon o x i d a t i o n T h e MCD is v i r t u a ll y id e n tic a l 2- in form and magnitude with the MCD of 4-Fe c lu s te r a t C o xid atio n le v e l, p a r tic u la r ly the MCD peak ju s t below 1000 nm which is 2_ diagnostic fo r the presence of a [ ^ 6 4 8 4 ( SR)4 ] c lu s te r . The large change in CD in the v is ib le range also in d ic ates a change in conformation of Av2 on o x id a tio n . This is a d ir e c t evidence of conformational change of Av2 in the presence of ATP as suggested by 68 ----------------------------gg gg----------------------------------------------------------------------------------------------------- other methods. * (see Chapters 6 -7 ) 3 .7 . Conclusion The absorption, CD, and MCD o f nitrogenase components from Av and Kp have demonstrated th a t the metal c lu s te rs and th e ir protein environment are s tr u c tu r a lly id e n tic a l. The CD and MCD are con sistent with find ings from other techniques in suggesting the absence of conventional 4Fe-S c lu s te r in FeMo p ro te in . The absorption, CD, and MCD of reduced and oxidized Fe proteins c h a ra c te riz e the presence of a 3 - 2- 4Fe-S c lu s te r changing from C to C s ta te during enzyme tu rn o ver. D ire c t evidence fo r a change in conformation of Av2 in the presence of ATP was shown by the CD, as i t is changed in form and magnitude in the v is ib le wavelengths range with the ad d itio n of ATP. 69 Figure 3 -1 . Diagram of the Gas-Tight Sample Cell Holder (Medial Section and Front View ). The c y lin d ric a l c e ll holder was made of anodized brass. The c ir c u la r window were made with fuzed quartz and sealed by double O -rin g s . The c e ll was held in place by a sample boat (see Figure 7 - 1 ) . The c e ll holder and sample c e ll boat were designed by F. D e v lin . 1.054 MEDIAL SECTION A-A .840 s r SAMPLE bom 5A M P Lfc-v r/ CELl^g % . . a $ I D , .8I60.D.AND .062 5 WIDE O-RING. 3^.551 I.D.,.691 O.D.AND.0625 WIDE O-RINGS. 2 .ALL O-RINGS GROOVES SHOULD BE .083 WIDE AND .050 DEER I. ALL DIMENSIONS ARE IN INCHES. MATERIAL IS BRASS. RETAINING RING WINDOW .7SX.0G25 ^-O-RING HOLDER Figure 3 -2 . Absorption ( a ) , CD ( b ) , and MCD ( a t 1 Tesla) (c) Spectra of Reduced Av1 (----) and Kp1 (----- ) . The pro tein samples were in 0.025 M HEPES, pH 7 .4 / 0.25 M NaCI (Av1 in H2 0) or 0 .50 M NaCI (Av1 in D2 0; Kp1 in H20 or D2 0 ) . Samples were in ^ 1 .6 m M d ith io n it e . Av1 and Kp1 concentrations were less than 0.25 mM. 72 300 aooo IQOO A« 1 0 - 50 50,000 5 POO 20 30 73 Figure 3 -3 . Absorption ( a ) , CD ( b ) , and MCD (a t 1 Tesla) (c) Spectra of Reduced Av2 (------) , Reduced Kp2 and P o st-S tead y-S tate Oxidized Av2 ( ------) . Reduced Av2 and Kp2 samples were in 0.025 M HEPES, 0.1 mg/ml DTT, 2 m M MgC^* and 2-4 m M d ith io n it e , pH 7 .0 - 7 .4 . Oxidized Av2 samples contained (mole/mole) Av1 , 0.1 mg/ml DTT, 12 m M MgCI2 , 10 m M ATP, 20 m M c re a tin e phosphate, and 1 mg of c re a tin e phosphokinase. D ith io n ite exhaustion was determined s p e c tro s c o p ic a lly . Av2 and Kp2 concentrations were less than 1 mM. 74 sz. Ot 02 0 0 0 *0 1 0 0 0 * 1 7 0 5*0 - ro O 'l T O R t o o T CHAPTER 4 MEDIUM AND TEMPERATURE EFFECTS ON REDUCED AV2 4 .1 . Introduction CD and MCD of reduced Av2 have been shown to be observable across 92 a wide spectral range (3 0 0-2,000 nm). The absorption and MCD r e f le c t the metal chromophore s tru c tu re and are in s e n s itiv e to the p ro tein environment, w hile the natural CD in the v is ib le and near u lt r a v io le t range is a ffe c te d by changes of p ro tein conformation around the chromophore. This chapter describes the use of CD to examine external p ertu rb atio n s on solutio n s of d ith io n ite -re d u c e d Av2. The pertu rb atio n s include changes in ionic s tre n g th , pH, type of b u ffe r, and tem perature. 4 .2 . Experimental 4 .2 .1 . General Preparation of P ro tein Samples Av2 was p u rifie d as described in Chapter 2. All p ro tein samples fo r spectroscopy were prepared from a p u rifie d batch of Av2 (800 mg of Av2 a t 20 m g/m l), frozen in 0.025 M T r is , 2 m M M gC^, 0.1 mg/ml DTT, 1-3 m M DT, a t pH 7 .4 . The Av2 enzyme, c r o s s -titr a te d with 76 s e m i-p u rifie d Av1 (F e M o -ll), has s p e c ific a c t iv it y of 1 ,5 0 0 -1 ,7 0 0 . B uffer solution s and protein s were d ith io n ite -re d u c e d and handled inside of a glove-box. The box was continuously flushed with R id o x -p u rifie d N^. The d iff e r e n t spectroscopic samples were prepared in advance and stored in liq u id n itro g e n . They were thawed under Ar fo r spectroscopy measurements. Exchange of b u ffe r so lu tio n in Av2 were done over PM-30 membrane in an Am icon 8 M C d i a f i I t r a t i o n c e l l , pressurized by deo xo-purifie d a t 10-35 p s i. Complete exchange of b u ffe r solu tio n (^ 9 9 % exchanged) took 3-5 hrs. During th is period of tim e, the c o lle c te d volume of f i l t r a t e exceeded 5x the fin a l volume of enzyme. The reducing power of f i l t r a t e and p ro tein was v e r ifie d with methyl viologen (Koch-Light Labs., England). 4 .2 .2 . V a ria tio n of lonic-S tren g th The ionic strength of b u ffe r was varied by adding increasing amounts of NaCI. Three ml of Av2 were concentrated 3 -fo ld over PM-30 membrane and exchanged into 0.025 M HEPES (Calbiochem, U ltro l g rad e), 2 m M MgC^ ( Mai I in ckro d t, AR grad e ), 0.1 mg/ml DTT (B io -R ad ), 2 m M DT (Matheson, Coleman & B e l l ) . The concentrated Av2 was d ilu te d with HEPES b u ffe r to 3 ml and divided in 0 .7 ml portions into 3 te s t tubes which contained 1 0 .2 , 1 4 .3 , and 23.0 mg of dry NaCI (Mai Iin c k ro d t, AR grade) to make fin a l solution s of Av2 in 0 .2 5 , 0 .5 , and 0.75 M NaCI re s p e c tiv e ly . The samples were stored frozen in a liq u id nitrogen c o n ta in e r. 77 4 .2 .5 . V a ria tio n o f pH The Av2 enzyme was exchanged into b u ffe r a t four d iff e r e n t pH. Three samples of pro tein were exchanged into 0.025 M HEPES/DTT/MgCI^/DT a t pH 6 .8 , 7 .4 , and 8 .0 re s p e c tiv e ly . The enzyme so lu tio n a t pH 8.5 was exchanged in T ris b u ffe r. The pH of b u ffe r solution s and enzyme were measured inside of the glove-box with a Beckman Futura electro d e 4 m m in diam eter, capable of measuring less than 50 yl of a liq u o ts . 4 .2 .4 . V a ria tio n of B uffer Besides HEPES and T r is , Av2 was exchanged into phosphate b u ffe r. The phosphate b u ffe r was prepared by mixing 1:4 mono/dibasic phosphate (MaI Iin c k ro d t) and t it r a t e d to pH 7 .4 with monophosphate. The phosphate b u ffe r contains 0.025 M Phosphate, 2 m M MgC^* 0.1 mg/ml DTT, and 2 m M DT. 4 .2 .5 . V a ria tio n of Temperature The tem perature of the p rotein so lu tio n was varied over the range 5-40°C . The c e ll and c e ll holder was cooled and heated sim ultaneously inside of a c y lin d ric a l ja c k e t. Water from a thermostated bath, which c irc u la te s through the ja c k e t, was cooled to 4°C with ice and slowly heated to 4 0 °. The tem perature inside of the c e ll was monitored a e ro b ic a lly in a te s t-ru n with copper-constantane thermocouples, and was found to be equal to the tem perature of the water bath. The tem perature of the water bath was th e re fo re assumed to be the 78 tem perature of the pro tein s o lu tio n during spectroscopic measurements. CD of reduced Av was measured a t 5 , 10, 20, 30, 40, and back to 30° re s p e c tiv e ly . The two spectra a t 30° should be superimposabIe to ensure re v e rs ib le tem perature e ffe c ts . 4 . 2 . 6 . Spectroscopic Measurements Absorption spectra over the 200-800 nm range were recorded with a Cary 17 spectrophotom eter. CD spectra were measured by a Cary 61 sp e ctro p o la rim eter. The m olecular e x tin c tio n c o e ffic ie n t e and d if f e r e n t ia l e x tin c tio n c o e ffic ie n t Ae were c a lc u la te d using Av2 m olecular weight of 6 4 ,0 0 0 . Suprasi! c e ll with 5 m m pathlength were f i l l e d with Av2 a t 20-22 mg/m I . The c e ll was enclosed in a g a s -tig h t holder described in Figure 3 -1 . The OD o f the p ro tein sample over the v is ib le -n e a r UV range increases from 0 .2 to 2 .0 0 . Most spectra were recorded a t least tw ice a t 1-3 hr in te rv a ls to ensure tim e-independent r e s u lts . A c tiv ity assays, b iu re t d eterm in ation, and pH measurements were done p rio r and subsequent to spectroscopy. The ^ 2 ^ 2 ac" * ’ ’v ^ V ° * a ll Av2 samples were >90/6 retain ed a fte r 3-5 hr of spectroscopic stu d ies. 4 .3 . Results and Discussion Figure 4 - 1 ,2 ,3 , and 4 show the CD AR (a n is o tro p ic r a t io As/s) spectra of d Ith io n ite -re d u c e d Av2, subjected to medium and tem perature changes. The CD changes were expressed in terms of a n is o tro p ic r a t io AR, ra th e r than d if f e r e n t ia l e x tin c tio n c o e ffic ie n t As , because of its 79 independence on concentration and c e ll path length, providing more s e n s it iv it y and accuracy. Like with reduced Av1 ^ e m ecjium and tem perature p erturbatio ns studied presented no s ig n ific a n t e ffe c ts on the v is ib le and near UV CD of reduced Av2. W ithin the lim its of experimental e rro rs , the re s u lts in d ic ate no major p ro tein conformation change a t the metal chromophore of Av2. Such r i g i d i t y of the p rotein moiety in the p ertu rb atio n s covered contrasted with fin d in g s by other methods. The dependance of ESR parameters on pH were observed in Cp2 in the presence of A T P . ^ Furthermore, in view of a s ig n ific a n t d is c o n tin u ity below 20°C in Arrhenius p lo ts for 102 nitrogenase catalyzed reductions, and the changes in Kp1:Kp2 association constant a t 17°C ^^ which suggest a conformational change in e ith e r FeMo or Fe p ro te in , the v is ib le -n e a r UV CD AR spectra at 5°-40°C in d ic a te no major change in reduced Av2 p ro tein s tru c tu re . The e ffe c ts of b u ffe r types (e .g . amine vs phosphate) and ionic strength ( e .g . increasing NaCI concentrations) on reduced Fe p ro tein have not been previously stu d ied . The p ro tein s tru c tu re of reduced Av2 remains independent of b u ffe r ty p e . ( i . e . T r is , HEPES, and phosphate) and ionic strength (0 , 0 .2 5 , 0 .5 0 , and 0.75 M NaCI in HEPES), as shown by the AR spectra in the v is ib le and near UV region (F ig ure 4 - 1 ) . 80 Figure 4 -1 . CD AR Spectra of Reduced Av2 as a Function of NaCI C oncentration. The p ro tein samples were in 0.025 M HEPES, 0.1 mg/ml DTT, 2 m M MgC^* and <_2 M m d ith io n it e , pH 7 .4 . Av2 concentrations were less than 20 mg p ro te in /m l. a) o 0 M NaCI; b) A 0.25 M NaCI; c) o 0 .5 M NaCI; d) • 0.75 M NaCI. The v a ria tio n s between the AR spectra were caused by experimental e rro rs . 0 0 A e / e xio4 o .0 o 400 500 700 600 x (nm) 00 N > Figure 4 -2 . CD AR Spectra of Reduced Av2 as a Function of pH. Av2 samples a t pH 6 .8 , 7 .4 , and 8 .0 were dissolved in 0.025 M HEPES, 0.1 mg/ml DTT, 2 m M MgC^* and < 2 m M d it h io n it e . Av2 sample a t pH 8 .5 was in 0.025 M T r is , 0.1 mg/ml DTT, 2 m M MgC^ and 2 m M d it h io n it e . The p ro tein concentrations were less than 30 mg/ml. a) A 6 .8 ; b) o 7 .4 ; c) a 8 .0 ; d) • 8 .5 . The v a ria tio n s between the AR spectra were caused by experimental e rro rs . 83 -d - 00 00£ (UlU) Y 009 O O S O Q tr 0 7 - 01- v0 l* '3 /3 - V 0*1 0 7 Figure 4 -3 . CD AR Spectra of Reduced Av2 as a Function of B u ffe r. The p rotein samples contained less than 20 mg p ro te in /m l, 0.1 mg/ml DTT, 2 m M MgC^* and < _ 2 m M d it h io n it e , pH 7 .4 . B u ffer concentrations were 0.025 M. a) o HEPES; b) A T r is ; c) u phosphate. The v a ria tio n s between the AR spectra was caused by experimental e rro rs . V O C O 00/ (U IU ) x 009 005 OOfr O 'Z- O ' I" ^oi* 3/ 3V oi O'z Figure 4 -4 . CD AR Spectra of Reduced Av2 as a Function of Temperature. Av2 p ro tein was in 0.025 M HEPES, 0.1 mg/ml DTT, 2 m M MgC^# and 2 m M d ith io n it e , pH 7 .4 . The p ro tein concentration was less than 35 mg/ml. a) A 5°C; b) • 10°C; c ) d 20°C; d) A 30°C; e) o 40°C. The v a ria tio n s between the AR spectra were caused by experimental e rro rs . 00 C O (U iU ) Y ooz 009 OOS o-z- 0' I’ ^01* 3 / 3 V 01 O'Z J CHAPTER 5 FE ION CHELATION AND CD STUDIES OF AV2 PROTEIN 5 .1 . Introduction Because of its extreme s e n s it iv it y to 0 ^ , the p u rifie d Fe component is often contaminated with appreciable amounts of the in a c tiv e or denatured form. This could lead to e rro rs in measuring the e x tin c tio n c o e ffic ie n t o f Fe p ro tein or other a n a ly tic a l work where accurate value of a c tiv e Fe enzyme concentration is essential (determ ination of MgATP binding con stant, k in e tic s s tu d ie s ). Studies on the e x tra c tio n of Fe ions from Fe p ro tein of C lo s trid i u rn pasteurianum were found useful in estim ating the percentage of 98 99 98 in a c tiv e enzyme. * Walker and Mortenson used 2 , 2 f -d Ip y rid y I to ch e late F e(I I ) from the Fe component of nitrogenase. The authors noted an i n i t i a l rapid c h e la tio n , followed by a slow phase which can be accelerated by the presence of MgATP. Since MgATP enhances the ra te of tra n s fe r of Fe ions to c h e la to rs , i t was believed th a t the bindings of MgATP induce a change in conformation of the Fe p ro tein causing the iro n -s u lfu r center to become more accessible to the ch e la tin g agents. The i n i t i a l c h e la tio n , observed in the absence of MgATP, was a ttrib u te d to the formation of complex between the chelato rs and the Fe ions from denatured Fe p ro te in . 89 99 In la te r s tu d ies, Ljones and B u rris Introduced bathophenathrolIne d ls u lfo n a te (BPD) as ch e la tin g agent fo r Fe ions from c lo s t r id ia l Fe enzyme. BPD was indicated to have higher ch e latin g power, more aqueous s o lu b ilit y , and a higher value fo r emax of the Fe(I I ) complex than 2 , 2 * - d ip y r i d y l . The authors reported th a t only 57$ of Fe atoms from Fe p ro tein with acetylene SA o f 1700 were chelated by BPD in the presence of MgATP. The re s t of the Fe ions believed to belong to in a c tiv e enzyme were chelated ra p id ly by BPD before the ad d ition of n u c leo tid es. By e x tra p o la tio n , the f u ll y a c tiv e c lo s t r id ia l Fe enzyme would have acetylene SA of 2980. Both 2 ,2 * -d ip y r id y I and BPD do not re a c t with the FeMo component e ith e r in 98 99 the presence or absence of MgATP. 9 As discussed in the previous ch a p te r, the v is ib le and near UV CD of Fe pro tein is sensible to the p ro tein environment of the metal chromophore. I t is th e re fo re of in te re s t to use CD as a n a ly tic a l tool fo r the assays of in a c tiv e Fe p ro te in . A s id e -to -s id e studies of BPD ch e latio n and CD as a function of enzymic a c t iv it y o f p a r tia l and complete (^-damaged Av2 p ro tein is presented in th is ch ap ter. 5 .2 . Experimental 5 .2 .1 . C ontrolled 0^- In a c tiv a tio n of Av2 The Av2 enzyme used has s p e c ific a c t iv it y of 1700-1900 nmoles of ethylene produced per min per mg of enzyme. Av2 was dissolved in 0.025 M HEPES, 2 m M MgCI2 , 0.1 mg/ml DTT a t pH 7 .4 . Frozen Av2 was 90 thawed In serum-stoppered 2 dram -slze v ia l which was dearated with Ar. D ith Io n ite was added to the thawed enzyme at 2% v /v . Next the serum stopper was removed and the v ia l g en tly shaken In a ir for In a c tiv a tio n . Samples of in a c tiv a te d Av2 were withdrawn a t each desired air-exp o su re tim e , in jected In 250 yl amounts to Ar-flushed v ia ls , and Immediately reduced with 2% v /v of d it h Io n it e . The enzyme samples were stored under Ar pressure during a c t iv it y and spectroscopic measurements. Av2 was successively exposed to a ir for 0, 1, 2 , 4 , 6 , and 30 min re s u ltin g in 0 , 30, 44, 77, 94, and 99% r e la t iv e loss of a c t iv it y re s p e c tiv e ly . The lig h t brown unexposed Av2 became dark brown as d ith io n ite and enzyme were ra p id ly oxid ized by a i r . P rotein p re c ip ita te d when exposed in a ir fo r more than 1 hour. The a ir-o x id iz e d enzymes turned lig h t yellow when reduced by d ith io n it e . The color o f reduced Av2 becomes lig h te r as the loss of a c t iv it y increases. A c tiv ity assays were done immediately p rio r to spectroscopic measurements. P ro tein concentration was determined by the b iu re t methods. 5 .2 .2 . C helation of Fe Ions from Av2 by BPD Bathophenanthroline d is u lfo n ic acid were purchased from A ldrich (New Jersey) and prepared in 1 m M so lu tio n in 0.025 M HEPES, 2 m M M gC^, and 0.1 mg/ml DTT. The c o lo rle s s so lu tio n of BPD was t it r a t e d to pH 7 .4 with 1 N NaOH. ATP (Sigma) was prepared In 0 .5 M so lu tio n which contains 0.025 M HEPES, 2 m M MgC^* and 0.1 mg/ml DTT, and was t it r a t e d to pH 7 .4 with 1 N NaOH. 91 D ith io n ite reagent was prepared in 0.08 M so lu tio n as described in 2 .3 . Both BPD and ATP so lu tio n s were deaerated with Ar and reduced by 2% v /v of d ith io n it e . Spectroscopic samples were handled a n a ero b ically inside of a Beckman c y lin d ric a l quartz c e ll w ith outer diameter of 21 m m and 10 m m lig h t path, sealed by double serum stoppers and a 3-way p la s tic v a lv e . The 3-way valve was m odified to perm it the opening o f 3 channels a t one time (see Figure 5 - 2 ) . The valve p osition s fo r d ea e ra tio n , sample In je c tio n , and spectroscopic measurements were described in Figure 5 -2 . The dearated cel I was f i r s t f i l l e d with 2 .5 m M of d ith io n ite -re d u c e d BPD, next is the in je c tio n of 100 yl of Av2, and la s t is 5 yl of ATP. The c e ll was shaken to allow reagents to mix thouroughly. The fin a l concentrations of each reagent are: 1 m M BPD, 0.083 m M Av2, 1 m M ATP, 2 m M MgCI2 , 0.1 mg/ml DTT in 0.025 M HEPES b u ffe r a t pH 7 .4 . The 0D a t 535 nm, corresponding to the e max o f Fe(I I ) BPD complex, were recorded with a Beckman Acta IV spectrophotometer. was measured Immediately fo llo w in g each ad d itio n of reagent. BPD turned pink in the presence of Av2. The add ition of MgATP caused the so lu tio n to turn dark pink. The corresponding spectroscopic e ffe c ts are id e n tic a l to the ones shown in referen ce 99. The presence of d ith io n ite was v e r ifie d before and a fte r spectroscopic measurements with methyl violo g en . The concentration of chelated Fe ions was c a lcu lated using £ 5 3 5 of 22140 M ^ cm ^ fo r the comp I ex The background absorption of a control sample containing a I I reagents except Av2 was substracted from the ATP-specific increase In 92 absorption. The number of Fe equ ivalen ts ( ie the mole ra tio n of Fe chelated in the presence of ATP per Av2 dimer) was p lo tte d against the resp ective SA of Av2 (Fig u re 5 - 3 ) . 5 .2 .3 . CD of 0 p ~ in a c t?vated Av2 Av2 was exposed to a ir as described in 5 .3 .1 . The protein was concentrated before a ir -in a c t iv a t io n to give concentration of 26.4 mg/ml. Enzyme samples were tra n s fe rre d to a ^ -f lu s h e d glove-box to f i l l c y lin d ric a l quartz cel Is of 2 m m or 5 m m path length. The c e lls were removed from the glove-box inside of g a s -tig h t holders. The absorption and CD were measured r e p e t it iv e ly to ensure tim e-independent sp e ctra . CD accross the 800-350 nm range was measured with a JASCO Model J-500C s p e c tro p o la rim e te r. The absorption was measured by Cary 17 spectrophotometer. A c tiv ity assays were done p rio r to spectroscopic measurements. The molar e x tin c tio n c o e ffic ie n t e, the molar d if f e r e n t ia l e x tin c tio n c o e ffic e n tA e , and the anisotropy r a t io AR Ae/e a t 440, and 400 nm were p lo tted against SA values. 5 .3 . Results and Discussion The ra te of a i r - i n a c t i v a t ion of Av2 is exponential as shown by the in a c tiv a tio n tim e course (F ig u re 5 - 1 ) . The h a l f - l i f e of d ith io n ite -re d u c e d Av2 in a ir is approxim ately 15 min. As shown by Table 5 -1 , the number of Fe eq u ivalen ts chelated by BPD in the absence of MgATP increases, w hile the number of MgATP-specific Fe equ ivalents decreases in conjunction with the decrease in s p e c ific a c t iv it y of 93 Av2. The amount of MgATP-specific Fe eq u ivalen ts tra n s fe rre d to BPD is proportional to the s p e c ific a c t iv it y of Av2 as shown by the lin e a r p lo t of Fe equ ivalen ts vs SA (F ig u re 5 - 3 ) . Assuming the Fe ions chelated in the presence of MgATP belong s o le ly to the a c tiv e form of Av2, and th a t th ere are 4 Fe ions per Av2 dimer,^ e x tra p o la tio n of the p lo t gives SA of 3,000 fo r f u ll y a c tiv e Av2. The SA o f f u ll y a c tiv e Av2 could be h ig h er, i f the lin e a r curve is s h ifte d to in te rs e c t the zero p o in t. This discrepency between the zero p oint and the other points on the p lo t could be a ttrib u te d to the contamination of non-enzymic Fe ions, a v a ria b le fa c to r which cannot be e a s ily c o n tro lle d or corrected for a n a ly t ic a lly . In s im ila r s tu d ie s , Ljones 98 and B u rris suggested SA of 2980 fo r f u ll y a c tiv e Fe p ro tein from C. pasteur ? anum. The general spectral featu res of the absorption and CD were conserved in the d iff e r e n t 0 ^ - in a ctiv ate d Av2 samples. They remained e s s e n tia lly id e n tic a l in form to the CD o f the control Av2 sample which was not exposed to a i r . The magnitude of the absorption and CD of Av2 decreases in conjunction with the loss in a c t i v it y , suggesting p rotein metal loss. D ras tic decreases in molar e x tin c tio n c o e ffic ie n t e and d if f e r e n t ia l molar e x tin c tio n c o e ffic ie n t Ae at 440 and 400 nm accompany severe loss of a c t iv it y ( i e more than 50% lo s s ), w hile the changes were moderate when the loss in a c t iv it y is sm all, as shown by the p lo t of e vs SA and Ae vs SA (F ig u re 5 -4 ) a t 440 and 400 nm. In c o n tra s t, the an iso tro p ic r a t io AR a t 440 and 400 nm remained r e la t iv e ly constant irre s p e c tiv e of the changes in s p e c ific a c t iv it y , as shown by the p lo t of AR vs SA (F ig u re 5 - 4 ) . 94 TABLE 5-1 . BPD CHELATI ON OF FE IONS FROM AV2. a b Min in SA Fe equ ivalen ts R e la tiv e a ir -ATP +ATP % loss of a c t iv it y 0 1755 0 . 2 2 2.50 0 1 1230 0.74 1 .89 30 2 979 0.97 1 .63 44 4 397 1 .44 1 . 0 0 77 6 1 0 0 1 .96 0.45 94 30 13 2.29 0 99 a. S p e c ific a c t iv it y = nmoles of 0 ^ ^ produced per min per m g of Av2 b. Mole r a t io of Fe ipns chelated per Av2 dimer 95 Figure 5 -1 . Time Course of Av2 A ir -In a c tiv a t io n . S p e c ific a c t iv it y (nmoles of C2 ^ reduced per minute per m g of p ro te in ) of Av2 were p lo tte d as a function of tim e in te rv a ls exposed in a i r . 96 2000 1000 0 9 5 3 7 Minutes Exposed in Air VO Figure 5 -2 . Valve P o sitio n s During the Spectroscopic Studies of BPD Fe C helation of Av2. A. Degas of 1 cm lig h t path c y lin d ric a l quartz c e l l . B. Sample in je c tio n under flushing Ar. C. Sample c e ll sealed fo r spectroscopic measurements. 98 pum p Ar -> ■ A. Degas B. Sample Injection C. Sealed Sample Cell v O v O Figure 5 -3 . Fe Equivalents Chelated by BPD in the Presence of MgATP as a Function of Av2 S p e c ific A c tiv ity . The number of Fe eq u ivalen ts chelated by BPD in the presence of ATP is lin e a r ly re la te d to s p e c ific a c t i v it y . E x trap o latio n to 4 Fe e q u iv a le n t (p t a) gives SA ^3000 fo r f u ll y a c tiv e Av2. 100 F e Equivalents 4.0 3.0 2.0 1 .0 500 1500 2500 S A (nm ethylene produced per min per m g of protein) o Figure 5 -4 . P lo ts o f e, Ae , and Ae/e as a Function of Av2 SA. a) o a t 440 nm b) A a t 400 nm The p rotein concentration was 26.4 mg/ml. Av1 was dissolved in 0.025 M HEPES, pH 7 .6 which contains 2 m M MgCI2 , 0.1 mg/ml DTT, and less than 2 m M in d ith io n it e . 102 4 Ae /e x 10 .0 .2 .8 .4 Ae .0 .0 3 2000 1500 1000 500 103 CHAPTER 6 AV2 "SELF-OXIDATION" 6 .1 . Introduction The Fe component of nitrogenase is known fo r its d isrep u tab le s e n s it iv it y to oxygen. Since sub stantial C ^ -in a c tiv a t ion leads to d iff e r e n t spectroscopic re s u lts (see Chapter 5 ) , enzymic a c t iv it y was c a r e fu lly v e r ifie d during the i n i t i a l measurements of d ith io n ite -re d u c e d Av2. In a d d itio n , the spectra were scanned a t 1-2 hour in te rv a ls to ensure tim e-independent r e s u lts . During th is process, i t was noted th a t the excess d ith io n ite slowly disappeared with no concom ittant changes in CD. When the d ith io n ite has com pletely disappeared, a new and d iff e r e n t CD spectrum ra p id ly developed. At the same tim e , the absorption increased and presented a "390 mm" band, c h a ra c te r is tic of o xidized Av2. This new form of oxidized Av2 remained s ta b le fo r several hours w ithout loss of a c t i v it y . MgATP accelerated the o xidation process and replaced the CD with a spectrum q u a lit a t iv e ly s im ila r to th a t of Av1-oxidized Av2. The numerous attempts to e lu c id a te th is previously unreported property of Av2 - to which was given the name of " s e lf-o x id a tio n " fo r convenience - are described in th is chapter. 104 6 .2 . Experimental Av2 was p u rifie d from several d iff e r e n t batches of b a c te ria . The enzyme preparations contained Av2 with SA ranging between 1500-2000. They were homogeneous by gel e le c tro p h o re s is . Metal analyses by atomic absorption on some p u rifie d Av2 batches yielded > 3.7 Fe atoms per Av2 dimer and no d etectab le Mo atom. The enzyme was dissolved at high concentration (15-30 mg/ml) e ith e r in 0.025 M T ris or 0.025 M HEPES, both a t pH 7 .4 . All Av2 samples contained 0.1 mg/ml DTT ( d i t h i o t h r e i t o l , Bio Rad). MgCI2 (Mai I inckrodt) and d ith io n ite (MCB) were added a t various amounts depending on the conditions stu d ied . ATP, ADP, AMP, and GTP a t 98-9956 pure were purchased from Sigma. UTP and CTP were purchased from Becton-Dickinson and Co. They were prepared in concentrated so lu tio n s with 1 -1 . 2 x molar excess of MgCI2 , and buffered in 0.025 M HEPES, 0.1 mg/ml DTT, a t pH 7 .4 . The d iff e r e n t reagents were stored frozen a t 4 ° . They were thawed, degassed with Ar, and reduced by 1.6 m M of d ith io n it e . Each nucleo tide reagent was in je cte d a n a e ro b ic a lly at 1-5$ v /v to Av2 inside of an 0 2- f r e e glove-box. Formamidine s u lf in ic acid or FSA was purchased from A ld ric h . D ith io n ite in Av2 preparations was replaced by 2 m M of FSA by d i a f i I t r a t i o n on PM-30 membrane with Am icon 8 MC u n it. The exchange of reducing agent was done an a e ro b ic a lly inside of a N2-flu s h e d glove-box. The presence of FSA was v e r ifie d w ith methyl vio lo g en . 105 I't D it h io n it e - fr e e and Mg - f r e e Av2 samples were obtained by Sephadex G-25 chromatography. Av2 was concentrated 3x by u l t r a f i l t r a t i o n on PM-30 membrane before chromatography. The gel and e lu tio n b u ffers were c a r e fu lly degassed for several minutes to remove traces of oxygen. Enzyme was loaded to a 1x10 cm Sephadex G-25 column, eluted in 0.025 M HEPES, 0.1 mg/ml DTT, and 2 m M MgC^* pH 7 .4 fo r d it h io n it e - f r e e sample, and in 0.025 M HEPES, 0.1 mg/ml DTT, pH 7.4 ++ “ H* fo r Mg - f r e e sample. The Mg - f r e e Av2 sample was reduced with d it h io n it e . H£ evo lution and C2 H2 reduction by " s e lf-o x id iz in g " Av2 were analyzed by gas chromatography with Varian 1440 aerograph and Varian 485 in te g ra to r. ^ ^ 2 was in jected into the sealed con tainer of " s e lf-o x id iz in g " Av2 and incubated fo r several hours before gc a n a ly s is . EPR of Av2 samples were measured by a Varian E-12 spectrom eter, equipped with an Oxford Instruments ESR-9 4° c ry o s ta t. The EPR tubes were f i l l e d inside of a glove-box, removed from the glove-box, sealed w ith serum stoppers and stored frozen in liq u id nitrogen c o n ta in e r. The pH o f p rotein so lu tio n s were measured inside of a glove-box w ith a Beckman Futura e le c tro d e . The e le ctro d e has 4 m m diameter and is capable of measuring pH of 50 yl a liq u o ts . The absorption was measured by Cary 17 spectrophotom eter. The CD in the near U V -v is ib le range was measured with Cary 61 and JASCO J500C s p e ctro p o la rim eters. C y lin d ric a l quartz c e lls o f 5 m m and 2 m m lig h t paths were f i l l e d inside of a glove-box. They were enclosed in g a s -tig h t holders and removed from the glove-box fo r spectroscopic 106 measurements. Spectra were scanned r e p e t it iv e ly to fo llo w changes a t 1-3 hr in te rv a ls . The a c t iv it y and protein assays were done p rio r and subsequent to spectroscopy. 6 .3 . Fe P ro tein "S e lf-O x id a tio n " D ith io n ite -re d u c e d Av2 was found to undergo spontaneous o x id a tio n . The oxid atio n process can be v is u a liz e d in two phases. In phase I , d ith io n it e g rad u ally disappeared w ithout s ig n ific a n t changes in CD. In phase I I , when d ith io n ite was exhausted, as monitored by the decrease in absorption a t 315 mm, the CD was ra p id ly replaced w ith a new spectrum. At the same tim e, the absorption developed a "390" m m band, c h a ra c te r is tic of oxidized Av2. The v is ib le -n e a r UV absorption and CD of reduced, A v1-oxid ized , and " s e lf-o x id iz e d " Av2, are shown in Figure 6 -1 . The CD of " s e lf-o x id iz e d " Av2 is remarkably s im ila r to th a t of dye-oxidized Av2 (c f Chapter 7 ) . The spectrum remained s tab le fo r several hours denoting an e q u ilib riu m s ta te . The duration of phase I varied with the i n i t i a l concentration of d ith io n it e . Phase I I reached e q u ilib riu m in less than 10 hours. " S e lf-o x id a tio n " seemed abated in improved preparations containing highly a c tiv e Av2 (SA 1800-2000) but persisted in the presence of MgATP. MgATP was noted to a c c e le ra te " s e lf-o x id a tio n " , and j o in t l y transformed the CD of Av2 (F ig u re 6 - 2 ) . The v is ib le -n e a r UV CD of MgATP-induced " s e lf-o x id iz e d " Av2 is s im ila r in form with th a t of Av1-oxidized Av2. Recognizing th a t the binding of MgATP lowers the 107 105 reduction p o te n tia l of Av2, i t is conceivable th a t the n ucleotide can enhance " s e lf-o x id a tio n " . The e ffe c ts of MgATP to " s e lf-o x id a tio n ” were r e p e t it iv e ly observed in successive preparations of Av2 from d iff e r e n t batches of c e lls . The phenomenon seemed independent of p rotein con cen tratio n . Samples con taining as low as 5 mg/ml to as high as 40 mg/ml were noted to " s e lf-o x id iz e " in the presence o f MgATP. S im ila r MgATP e ffe c ts were observed with s e m i-p u rifie d Fe II (c f Chapter 2 ) , despite the presence of other pro tein im p u ritie s . Av2 a c t iv it y was n early 100$ retain ed a f te r several hours of 1 nfi " s e lf-o x id a tio n " . Christou e t aj_- reported re c e n tly th a t can be oxidized to C F e^S ^S P h)^^ by PhSH, y ie ld in g " S e lf-o x id iz e d " Fe p ro tein was assayed fo r H^ase a c t iv it y in view of th is re a c tio n . Using the absorption as m onitor, d ith io n ite -re d u c e d Av2 was le t to " s e lf-o x id iz e " under Ar pressure, in a sealed c u v e tte . The gas phase was gc analyzed before and a fte r " s e lf-o x id a tio n ." No t H2 was detected (th e assay s e n s it iv it y was 2 nmoles of despite the rapid disappearance of excessive amounts of d ith io n it e (p resen t a t 8 x molar excess with respect to Fe p r o te in ), and the presence of d i t h i o t h r e i t o l , a th io l a d d itiv e to a I I Av2 samples. ^2^2 ac'*'*v ^ y was s im ila r ly assayed over " s e lf-o x id iz in g " Av2. The lack of ^2^2 a c t i v it y , follo w in g " s e lf-o x id a tio n " made i t u n lik e ly to be caused by Av1 contaminants. " S e lf-o x id a tio n " was also observed in Kp2 s o lu tio n s in the presence of MgATP (work done by D. Lowe). The CD o f " s e lf-o x id iz e d " Kp2 was v i r t u a l l y id e n tic a l to th a t of " s e lf-o x id iz e d " Av2. Likew ise, 108 Cp2 was noted to undergo " s e lf-o x id a tio n ” in the presence of MgATP, but produced a d iff e r e n t CD spectrum. The ra te of ” s e lf-o x id a tio n ” was s im ila r in Av2, Kp2, and Cp2 p ro te in s . I t is possible th a t ”s e If-o x id a tio n ” is a general property of Fe p ro te in , even though previously unreported. 6 .4 . The Decomposition of D ith io n ite in ” S e lf-O x id iz e d ” Av2 I t was established th a t the disappearance of d ith io n ite in " s e lf-o x id iz in g ” Av2 was not due to Og# which could enter the c e ll sample during spectroscopy or was residual w ith in the g a s -tig h t c e ll holder. P a ra lle l experiments with co n tro ls were done with c e ll samples of Av2 & MgATP. The enzyme was loaded sim ultaneously to both c e lls inside of a "Vacuum Atmospheres” box containing less than 1 ppm and sealed inside of g a s -tig h t holders. Cell 1 was withdrawn from the box fo r spectroscopic measurements. Cell 2 was stored inside of the box as Q y- f r e e c o n tr o l. The control was removed from the box when sample c e ll 1 began to " s e lf- o x id iz e ” fo r comparative spectroscopy. The control was found to e x h ib it id e n tic a l CD with th a t of c e ll 1. D ith io n ite decomposes in so lu tio n a t ambient tem perature. The decomposition products caused sub stan tial pH change in control b u ffe r samples (0.025 M HEPES). In c o n tra s t, " s e lf-o x id iz e d ” Av2 only caused the pH to decrease from 7 .4 to 6 .8 , which e v id e n tly could not account fo r the accelerated decomposition of d ith io n it e . " S e lf-o x id a tio n ” occured both in d it h io n it e - f r e e Av2 and samples containing up to 25:1 molar excess of d ith io n ite to Av2, although the ra te o f 109 " s e lf-o x id a tio n " is n oticeab ly decreased in the presence of excess 2+ d ith io n it e . The removal of Mg by Sephadex G-25 chromatography did not a lte r the ra te of " s e lf-o x id a tio n " . " S e lf-o x id a tio n " did not cease when d ith io n ite is su b stitu te d by foram idine s u lf in ic acid (FSA). FSA was found to be useful as biochemical r e d u c t a n t j ^ and u n lik e d ith io n it e , can be obtained in the c r y s ta llin e s ta te . " S e lf-o x id a tio n " in the presence of MgATP occured a t the same ra te with FSA as with d ith io n it e . 6 .5 . S p e c ific ity of MgATP fo r "S e lf-O x id ize d " Av2 The s p e c if ic it y of the e f fe c t of MgATP on Cp2 has been 43 investigated in EPR measurements and a - a * - d ip y r iI ch e la tio n of Fe 98 ions. Various purine and pyrim idine nucleotides were te s te d . Of these te s te d , including 3 ,y-m ethylene ATP, ADP, AMP, GTP, UTP, and CTP, only the f i r s t two compounds e x h ib ite d comparable e ffe c ts , i f not weaker, to th a t of ATP. Figure 6 -3 , 6 -4 , and 6-5 show the e ffe c ts of ADP, AMP, GTP, CTP, and UTP on the v is ib le -n e a r UV CD of reduced and " s e lf-o x id iz e d " Av2. Only MgADP caused and e ffe c t comparable with th a t of MgATP. " S e lf-o x id a tio n " was eq u ally accelerated by MgATP, g ivin g id e n tic a l CD spectrum. Other nucleotides were in a c tiv e . " S e lf-o x id iz e d " CD was n eith er accelerated nor m odified by these compounds. Furtherm ore, reduced Av2 CD was found unsensitive to these nu cleo tid es. 110 6 .6 . EPR of "S elf-O xid I zed” Av2 The extent of " s e lf-o x id iz e d ” Av2 CD, e ith e r w ith or w ithout MgATP, appeared s u b s ta n tia lly sm aller than th a t of dye-oxidized Av2 or Av1-oxidized Av2. Q u a n tita tiv e comparison suggested incomplete * ox i d a tio n . The EPR measurements were found in accord with these re s u lts . Reduced Fe p rotein presents an EPR signal near g = 2 , s im ila r to th a t 108 43 44 of spinach fe rre d o x in , o xidized Fe p ro tein has no EPR. ' MgATP was found to a lt e r the EPR spectrum of reduced Fe p ro te in , suggesting 1 no bindings and a change in conformation of the p ro te in . The EPR measurements of reduced and " s e lf-o x id iz e d " Av2, e ith e r with or w ithout MgATP present, were done at 13°K. The EPR tubes were f i l l e d inside of a glove-box and ra p id ly frozen in liq u id n itro g e n . Reduced Av2 samples were immediately frozen follow ing the a d d itio n of MgATP and excess d it h io n it e , to avoid " s e lf-o x id a tio n " . " S e lf-o x id iz e d " samples were monitored by CD before in je c tin g in to the EPR tubes. The EPR spectra of reduced and " s e lf-o x id iz e d " Av2 are shown in Figure 6 - 6 . They are id e n tic a l in form, but d if f e r in magnitude. The EPR of " s e lf-o x id iz e d " Av2 is considerably sm aller than th a t of reduced Av2. S im ila r re s u lts were obtained with MgATP present. The bindings of MgATP changes the EPR spectrum as shown in Figure 6 -7 . The EPR spectrum of " s e lf-o x id iz e d " Av2 is much sm aller than th a t of reduced Av2, both with MgATP presen t. MgADP also presented s im ila r q u a n tita tiv e e ffe c ts to the EPR of " s e lf-o x id iz e d " Av2, as shown in Figure 6 - 8 . 111 The EPR and CD re s u lts indicated th a t sub stantial amounts of reduced Av2 were s t i l l present a t e q u ilib riu m in "s e lf-o x ? d ize d ” Av2 samp Ie s . 6 .7 . Conclusion The re s u lts of the in v e s tig a tio n with " s e lf-o x id iz e d " Av2 can be summarized as fo llo w s: 1. " S e lf-o x id a tio n " is stim ulated by MgATP, MgADP. 2. The enhancement of " s e lf-o x id a tio n " is s p e c ific to MgATP and MgADP. 3. The phenomenon also occurs in Kp2 and Cp2 s o lu tio n s , suggesting th a t i t could be general with Fe p ro te in . 4. " S e lf-o x id a tio n " takes place e ith e r in the presence of absence of d it h io n it e . 5. The pH change accompanying the " s e lf-o x id a tio n " of Av2 is too small to account fo r the accelerated decomposition of d it h io n it e . 6 . The disappearance of d ith io n ite and the " s e lf-o x id a tio n " process are not caused by tra c e of e ith e r residual or leaking in to the samp Ie cel I . 7 . FSA, a s u b s titu te fo r d ith io n it e , does not abate " s e lf-o x id a tio n " . 8 . is not produced fo llo w in g " s e lf-o x id a tio n " . 9 . ^ 2 ^ 2 ' S n°^ re(*uced ^2^4 over ”s e * f ” o x ‘ d i z ing" Av2 samples. 10. The CD and EPR measurements shows th a t o xid atio n is not complete. 112 The s ig n ific a n c e of " s e lf-o x id a tio n " to the c a t a ly t ic mechanism of nitrogenase is not known. However, i t presents com plication to the studies of MgATP-reduced Fe p ro tein in te ra c tio n s . The phenomenon is in trig u in g and should remain to be e lu c id a te d . 113 Figure 6 -1 . V is ib le -n e a r UV Absorption (a) and CD (b) Spectra of Reduced (------) , Av1-Oxidized ( - • - • - ) , and "S e lf-O x id ize d " Av2( ) . Reduced and " s e lf-o x id iz e d ” Av2 samples contained 0.1 mg/ml DTT, 2 m M MgC^* and i n i t i a l l y 2-4 m M d ith io n it e . Av1-oxidized Av2 samples i n i t i a l l y contained \% (mole/mole) Av1 , 0.1 mg/ml DTT, 12 m M M gC^, 10 m M ATP, 20 m M c re a tin e phosphate, and 1 mg of c re a tin e phosphokinase. Av2 concentration were less than 0 .5 mM, in 0.025 M HEPES, pH 7 .0 - 7 .4 . 114 x (nm) 80C 500 400 2.0 C 30 3 10 10 15 20 25 30 V (xl O ’ ^cnT^) 115 Figure 6 -2 . V is ib le -n e a r UV CD Spectra of Reduced (------ ) and " S e lf-O x id iz e d ” Av2 ( ------) in the Presence of MgATP. Av2 samples were i n i t i a l l y in 0.025 M HEPES, 0.1 mg/ml DTT, 2 m M MgC^* and 2-4 m M d ith io n ite . "S e lf-O x id ize d " Av2 in the presence of MgATP contained 1.5 m M MgATP. Av2 concentrations were less than 0 .3 mM. 117 800 700 600 400 X (nm) 6.0 Ae Z 0 - 1.0 r \ i \ \ \ \ / \J 12 14 20 24 28 32 _____________________________v (x 10“ ^cm ^)________________________ __ Figure 6 -3 . V is ib le -n e a r UV CD Spectra of Reduced Av2 in the Presence of MgAMP (-----) , MgGTP (-------) , MgCTP (----------) , and MgUTP ( .............) . Av2 samples were in 0.025 M HEPES, 1 mg/ml DTT, 2 m M M gC^, and 2-4 m M d ith io n ite pH 7 .4 . MgAMP, MgGTP, MgCTP, and MgUTP concentration were ^10 mM. Av2 concentrations were less than 0 .3 mM. 118 119 x (nm) 800 700 600 500 400 A t: 7 / 0.2 0 ¥ • \ / \ v v / v \ / \ y 0.2 - 0 .5 v ( X 1 0 ' \ m ^ Figure 6 -4 . V is ib le -n e a r UV CD Spectra of "S e lf-O x id ize d " Av2 in the Presence of MgAMP (-----) , MgGTP (------- ) , MgCTP ( ---------) , and MgUTP ( ...........) . Av2 samples were in 0.025 M HEPES, 0.1 mg/ml DTT, 2 m M MgC^ i n i t i a l l y a t pH 7 .4 . MgAMP, MgGTP, MgCTP, and MgUTP concentrations were ^10 mM. Av2 concentrations were less than 0 .3 mM. 120 i {.m£ otx) a OE 9 Z 11 81 M 0 00 z 009 008 (uni) \ Figure 6 -5 . V is ib le -n e a r UV CD Spectra of Reduced (------ ) and "S e lf-O x id ize d " (-----) Av2 in the Presence of MgADP. Av2 samples were in 0.025 M HEPES, pH 7 .4 , 0.1 mg/ml DTT, 2 m M MgC^* and i n i t i a l l y ^ 2 m M d it h io n it e . MgADP concentration was less than M mM, Av2 concentra tio n s were less than 0 .3 mM. 123 400 500 600 700 800 X (nm) .0 \ J 0 0 .0 (x l0 ^cm Figure 6 - 6 . E ffe c ts of "S e lf-O x id a tio n " on the EPR of Av2. The EPR tubes contained Av2 (3 6 .2 mg p ro te in /m l) in 0.025 M HEPES, 0.1 mg/ml DTT, 2 m M MgC^* M .1 m M MgADP, and i n i t i a l l y pH 7 .4 . a) EPR spectrum of Av2 reduced by ^2 m M d ith io n it e . b) EPR spectrum of " s e lf-o x id iz e d " Av2. The s e ttin g s 2 of the instrument are: gain 2.5x10 , sweep ra te 100 G/min, tim e constant 0 .3 sec, microwave frequency 9.22 GHz, microwave power 2 .7 mW, modulation amplitude 6 .3 G, and tem perature 16°K. 124 200 __i_ ro VJTI g = 2.01 0 1 600 Gauss Figure 6 -7 . E ffe c ts of MgATP on the EPR o f Reduced and " S e lf-O x id iz e d ” Av2. The EPR tubes contained 0 .3 ml of Av2 (14 mg p ro te in /m l) in 0.025 M HEPES, 0.1 mg/ml DTT, 2 m M MgC^* and ^1 .5 m M MgATP. a) EPR spectrum of d ith io n ite -re d u c e d Av2+MgATP, d ith io n ite concentra tio n was less than 2 mM. b) EPR spectrum of " s e lf-o x id iz e d " Av2+MgATP. The s e ttin g s on the instrument are: gain 800, sweep ra te 250 G/min, tim e constant 0 .3 sec, microwave frequency 9.408 GHz, microwave power 9 mW, modulation amplitude 10 G, and tem perature 13°K. 126 127 g = 1 .978 9=2.07 g = l .955 q q r D g = 1 .896 100 1 50 Gauss Figure 6 - 8 . E ffe c ts of MgADP on the EPR o f Reduced and "S elf-O x id ize d " Av2. Av2 samples contained 14.6 mg protein/m l in 0.025 M HEPES, 0.1 mg/ml DTT, 2 m M MgCI2 , i n i t i a l l y pH 7 .4 . a) EPR spectrum of d ith io n ite -re d u c e d Av2+MgADP, the d ith io n ite concentration was ^2 mM. b) EPR spectrum of " s e lf-o x id iz e d " Av2+MgADP. The s e ttin g on the instrument were: gain 500, sweep ra te 500 G/min, tim e constant 0.03 sec, microwave power 9.405 GHz, microwave power 3 mW , modulation amplitude 10 G, and tem perature 13°K. 128 129 g = 2 .007 0 50 100 150 Gauss 1 -----1 -----1 _____ I CHAPTER 7 ATP AND ADP TITRATIONS OF DYE-OXIDIZED AV2 CD SPECTRA 7 .1 . Introd uction The in te ra c tio n of Fe p ro tein w ith ATP has been e x te n s ive ly 40-44 8 6 98 99 investigated by physical and chemical methods. ' ' The hydrolysis of ATP to ADP is o b lig a to ry to nitrogenase substrate reductio n . The s p e c if ic it y fo r ATP, in opposition to other purine and 43 pyrim idine n ucleo tides, and a , 3 -dim ethyIene analog o f ATP, im plicates th a t the adenosine a , 3 -diphosphate group is involved in binding. The y-phosphate is a d d itio n a lly e s s e n tia l, since the binding 86 2+ of ADP is in h ib ito ry to a c t i v it y . Furthermore, the presence of Mg 2"t* 2 + 2+ 2+ is required; other d iv a le n t cations such as Mn , Co , Fe , Ni are 9D 1 HQ less e f fe c tiv e s u b s titu te s . * 9 On the basis of EPR 43 44 19 t i t r a t i o n 9 and gel e q u ilib r a tio n , i t is c u rre n tly believed th a t 2 molecules of MgATP or MgADP bind to one Fe p ro tein dimer. The d isso c ia tio n constants of the nucleotides have been obtained by these 43 44 methods. As suggested by the EPR measurements, 9 and la te r 98 99 92 confirmed by the Fe ch elatio n studies 9 and CD spectroscopy, the binding of MgATP was noted to change the p ro tein s tru c tu re of the Fe component, but presented in s ig n ific a n t e ffe c ts to the conformation of 130 isolated FeMo component. The mechanism of ATP binding and its hydrolysis in nitrogenase substrate reduction remain obscure, a lb e it extensive in v e s tig a tio n . A ll previous studies involved Fe p ro tein in the reduced s ta te . L i t t l e is known about ATP and ADP in te ra c tio n s with oxidized Fe p ro te in . K in e tic s model s , ^ ^ have considered the in te ra c tio n of MgATP with oxidized Fe. One important discovery, in conjunction with the " s e lf-o x id a tio n " phenomenon, was th a t ATP has minor e ffe c t on the CD spectrum of reduced Av2 p ro te in , but caused pronounced changes to the CD of oxidized Av2. S im ila r c h iro p tic a l changes were observed with Av1-oxidized Av2 sample. Considering th a t the enzymic o xid atio n of the Fe component in fix in g system requires the presence of ATP, i t would be of in te re s t to pursue the studies of ATP:oxidized Fe p rotein in te ra c tio n s . Fe pro tein can be non-enzym atically oxidized using dyes. Recently, Lowe has found th a t indigo carmine oxidized Kp2 w ithout severe loss of a c t iv it y . The t i t r a t i o n of dye-oxidized Kp2 with ATP and ADP has yielded CD curves which perm it the determ ination of binding stoichiom etry and e q u ilib riu m constants. Av2 can also be oxidized by indigo carmine. The CD of dye-oxidized Av2 is d r a s tic a lly changed in the presence of ATP or ADP. This chapter presents the t i t r a t i o n of th is c h iro p tic a l e ffe c t w ith ATP and ADP and the determ ination of th e ir binding sto ich io m etries and d isso c iatio n constants. 131 7.2. Experimental The preparation of dye-oxidized Av2 was done inside of a high q u a lity "Vacuum Atmospheres" glove-box, f i l l e d with R id o x -p u rifie d r e -c ir c u la tin g He. The level inside the glove-box was less than 1 ppm. B uffers and reagents were e ith e r c a re fu lly degassed before entering the glove-box or prepared under the boxf s atmosphere. A ll reagents were buffered in 0.025 M HEPES, pH 7 .4 , which contained 0.1 mg/ml DTT, and 2 m M MgC^ (abbreviated HEPES/DTTT/MgC^ b u ffe r ). Indigo carmine (A n alar, U .K .) was prepared inside of the glove-box by dissolving ^5 m g of indigo carmine i n i ml of degassed HEPES/DTT/MgC^ b u ffe r. The concentration of indigo carmine was 2 0 mM. Adenosine 5 ’ -trip h o s p h a te disodium s a lt (abbreviated ATP, > 98% HPLC pure) was purchased from ICN ( Ir v in e , C a lifo r n ia ) . ATP was dissolved in HEPES/DTT/MgC^ b u ffe r and t it r a t e d to pH 7 .4 with 1 N NaOH. The concentration of ATP was >_9 mM. The ATP concentration was determined by the absorption a t 259 nm, using molar e x tin c tio n c o e ffic ie n t eocn of 16x10^. 259 Adenosine 5 ’ -diphosphate sodium s a lt (abbreviated ADP, 95-99$ pure) was purchased from Sigma (S a in t Louis, M is s o u ri). The ADP reagent contained less than 21 m M ADP, dissolved in HEPES/DTT/MgC^ b u ffe r a t pH 7 .4 . The" concentration of ADP was measured spectrop hotom etricaIly using molar e x tin c tio n c o e ffic ie n t £ 2 5 9 15.4x10^. Both ATP and ADP reagents were degassed with Ar and brought inside of the glove-box in serum-sealed v ia ls . 132 Chromatography with Sephacryl S-200 gel was found superior in separating dye from oxidized Av2, compared to Sephadex G-10 or G-25. Pharmacia Sephacryl S-200 was washed and suspended in HEPES/DTT/MgCI^ b u ffe r. The gel and elution buffer were degassed under Ar before entering the glove-box, and subsequently degassed for the second time once inside of the glove-box. The gel was packed inside of a 1x15 cm glass column. Highly active Av2 (C2 H2 SA>2000) was used for the t it r a t i o n experiments. The frozen enzyme was brought in the antechamber of the glove-box, insulated by a th ick p la s tic holder, th a t was cooled with liquid nitrogen. After of 15 min of evacuation in the antechamber, Av2 was let to thaw under the box’ s atmosphere. Each t it r a t i o n experiment used 0.5 ml of highly concentrated Av2 (<1 mM). Av2 was oxidized with 0.1 ml of 20 m M indigo carmine, corresponding to 4-5x molar excess of dye. The presence of excess dye caused the mixture to turn dark blue. The mixture was shaken for several minutes before loading to the Sephacryl S-200 column. The column was next washed with degassed HEPES/DTT/MgCI^ b u ffer. Three colored bands were separated. Yellow reduced dye remained near the top of the column, next came the excess blue dye and brown oxidized Av2 respectively. The oxidized Av2 was collected in three fra c tio n s . The most concentrated fraction was used for the t it r a t i o n experiments. The gel chromatography diluted the o riginal Av2 sample by a factor of 3. Oxidized Av2 is extremely la b ile and loses a c tiv ity when stored in Iiquid nitrogen. 133 The t it r a t i o n of ATP to oxidized Av2 were done in two methods. M u ltip le c e lls were used in method I. Each cell was in d ividu ally f i l l e d with oxidized Av2 that contained a fixed amount of ATP, corresponding to a point on the t it r a t i o n curve. Generating 10-15 t it r a t i o n points by th is method required several experimental days, since only 4 c e lls can be measured at one time. Furthermore, method I involved separate preparations of oxidized Av2 th a t could have d iffe re n t a c tiv ity and concentration. Method I I , introduced in later experiments, is consisted of sequential additions of ATP to a single oxidized Av2 sample c e l l . A c y lin d ric a l quartz cell of 11 m m ID and 5 m m lig h t path was p a r t ia lly f i l l e d with 410 X of oxidized Av2, and successively injected with variable amounts of ATP inside of the glove-box. The bubble created in the cell was masked by a cell boat equipped with an o ff-c e n te r o r ific e (Figure 7 -1 ). The cell contained a miniature s tir r in g bar th at can be magnetically rotated inside of the cell to permit mixing, following each addition of nucleotide. The cell was sealed inside of a gas-tig h t holder and removed from the glove-box for spectroscopic measurements. Method I I can provide 9-12 t it r a t i o n points in one single experiment on one oxidized Av2 preparation, thus more e ffe c tiv e than method I . The t it r a t i o n of ADP to oxidized Av2 was done via method II only. Indigo carmine oxidized Av2 suffered no loss of a c tiv ity . Furthermore, the enzyme retained fu ll a c tiv ity a fte r several hours of spectroscopy. The CD was measured with JASCO J500C spectropolari- meter. The absorption was measured with Cary 17 spectrophotometer. 134 7 .3 . ATP and ADP Bind Ozfdlzed Av2 The v isib le -n e ar UV CD spectra of dye oxidized Av2 with and without ATP and ADP present are shown in Figure 7 -2 . The CD of dye-oxidized Av2 resembles that of "s e lf-o x id ize d ” Av2. A d d itio n ally, like with enzymatically oxidized Av2 and "self-o xid ized " Av2, the presence of eith er ATP or ADP creates radical change to the CD spectrum, implicating substantial change in protein structure. The s im ila r itie s between the respective CD spectra indicate th a t the oxidation products are identical despite the difference in oxidative processes. The size of dye-oxidized Av2 CD is much larger than that of "self-oxidized" Av2, suggesting p artial oxidation in the la tte r . This is also consistent with the EPR resu lts. The e ffe cts of ATP and ADP to the CD of dye-oxidized Av2 c le a rly demonstrate that the nucleotides bind oxidized Av2. Sim ilar results were observed in the CD studies of ATP and ADP binding to oxidized K p 2 .^ “ * Previous studies of ATP or ADP:Fe protein interaction dealt solely with the reduced state of the enzyme. The EPR measurements have suggested the 40-44 binding of ATP to reduced Fe protein. in contrast, the effects of ATP or ADP to the CD spectrum of reduced Av2 are very small. I t is not clear that eith er the interaction of the nucleotide with reduced Fe protein is in sig n ifican t or the CD is less sensitive to structural change of reduced Fe protein. K inetic models in which ATP bind to 112 oxidized Fe protein have been proposed, but later on rejected in 99 the Fe chelation studies. The CD results demand a re-evaluation of previous studies of the interaction of Fe with protein. 135 7 .4 . Difference Between the Bindings of ATP and ADP to Oxidized Av2 The t it r a t i o n of the CD using ATP and ADP are shown in Figure 7-3 and 7-4 respectively. The t it r a t i o n with ADP c le a rly shows isobestic points at 330, 385, and 450 nm, indicating the presence of two discrete species: ADP-bound and ADP-free Av2. In contrast, the t it r a t i o n with ATP does not show the isobestic point at 450 nm and pseudo-isobestic points at 330 and 385 nm. The CD signal centered at 470 nm successively decreases and re-increases as a function of increasing amounts of ATP; whereas with ADP, the CD a t 470 nm increases monotonously with increasing amounts of ADP. The changes in CD at 360, 410 and 470 nm were plotted against ATP or ADP/Av2 mole r a tio . Figure 7-5 shows the difference between the CD change at 470 nm caused by the t it r a t io n s with ATP and ADP to oxidized Av2. The t it r a t i o n curve is V-shaped with ATP and nearly hyperbolic with ADP. The difference between the t it r a t i o n curves with ATP and ADP is less pronounced at 410 nm as shown in Figure 7 -6 . The t it r a t i o n curve at 360 nm (Figure 7-7) suggested that ADP binds more e ffe c tiv e ly to oxidized Av2 than ATP. The results shows s ig n ific a n t difference in binding mode between ATP and ADP to Fe protein. 136 7 .5 . Stoichiometries and Dissociation Constants of the Bindings of ATP and ADP to Oxidized Av2 The ATP and ADPsFe protein stoichiometry can be d ire c tly obtained from the CD t it r a t i o n curves. The ATP t it r a t i o n curve at 410 nm, shown in Figure 7-6, suggested a stoichiometric number of 1 .6 - 1 . 8 for the binding constant of ATP to Fe protein. Since the true number is believed to be the lower value re fle c ts the presence of inactive Fe protein in the preparation, which does not bind ATP. Based on the bathophenanthroIine t it r a t i o n of inactive Av2 protein (see Chapter 5 ), the t it r a te d Av2 with SA of ^2000 must contain nearly 35$ inactive enzyme. The t it r a t i o n number increases to nearly 2 when corrected for th is e ffe c t. The influence of enzyme a c tiv ity to the t it r a t i o n curve is shown in Figure 7-7. Saturation is attained e a r lie r with less active Av2 preparation. Curve A belongs to Av2 with SA M 400; curve B belongs to Av2 with SA ^ 2 0 0 0 . The t i t r a t i o n with ADP reached equilibrium at 1.4 ADP/Av2 mole r a tio , as indicated by the t it r a t i o n curves at three separate wavelengths: 360, 410, and 470 nm (Figures 7-5, 7-6 and 7 -7 ). The ADP/Av2 mole ra tio a t equilibrium also increases to nearly to 2 when corrected for the presence of inactive Av2 in the preparation. The binding mode of ATP and ADP to Fe protein is hyperbolic, as shown by Figure 7-5. This mode of binding contrasts the sigmoidal 99 model proposed by Burris. The t it r a t i o n curve was f it te d to computer-generated models which include 2 non-cooperative site s and 2 mutually cooperative s ite s . The model is rejected following the 137 fa ilu r e to f i t the experimental data. Dissociation constants can be determined by th is method. The ATP t it r a t i o n was successfully f it te d to the two non-cooperative sites model with a ll-o r-n o th in g spectral — 7 — 8 e ffe c ts . The dissociation constant for ATP and ADP are 10 -10 M based on preliminary f i t s of the t it r a t io n curves. 138 Figure 7-1. Front and Side Views of Sample Cell Boat Used for Sequential T itr a tio n . The 5 m m path length cell has a total volume of M 50A . I t was p a r t ia lly f i l l e d with 410 A of oxidized Av2 to allow for the addition of nucleotide during the t it r a t i o n experiments, thus creating a water bubble on top of the c e ll. The bubble was masked by an o ff-c e n te r o r ific e on the sample cell boat. The miniature s tir r in g bar was magnetically rotated to permit thourough mixing inside of the sample cell following each addition of ATP or ADP. 139 TOP VIEW WATER BUBBLE LIGHT PATH MINIATURE STIRRING BAR \ \ .OGEDIA. HOLE TO FACILITATE EXTRACTION) OF SAMPLE CELL VIEW A-A FRONT VIEW Figure 7-2. Visible-near UV CD Spectra of Dye-Oxidized Av2 (------), Dye-Oxidized Av2 + MgATP ( ), and Dye-Oxidized Av2 + Mg A DP (----------). Av2 was oxidized by 4-5x molar excess of indigo carmine. The dye was separated by a Sephacryl S200 column. The protein was in 0.025 M HEPES, pH 7 .4 , 0.1 mg/ml DTT, and 2 m M MgC^- The concentration of ATP was < _ 1 mM. The concentration of ADP was <_0.5 mM. The concentration of Av2 was less than 0.2 mM. 142 a (nm) 700 600 500 400 .0 0 N ». 0 .0 Figure 7-3. T itr a tio n of Dye-Oxidized Av2 CD with MgATP. Oxidized Av2 was in 0.025 M HEPES, pH 7 .4 , 0.1 mg/ml DTT, and 2 m M MgC^. ATP was added sequentially to give: a) 0 mM, b) 0.022 mM, c) 0.044 mM, d) 0.088 mM, e) 0.153 mM, f) 0.259 mM, g) 0.362 mM, h) 0.423 mM, i) 0.483 mM, j ) 0.582 mM, and k) 3.79 mM. The i n it ia l concentration of Av2 was 0.189 mM. The cell path length was 5 mm. 143 144 A A baseline — 1 » * 1 ^ 1 350 400 450 500 nm Figure 7-4. T itr a tio n of Dye-Oxidized Av2 CD with MgADP. Oxidized Av2 contained i n i t i a l l y 12 mg protein/ml in 0.025 M HEPES, pH 7 .4 , 0.1 mg/ml DTT, and 2 m M MgCI2 . ADP was added sequential in the following m M concentrations: a) 0, b) 0.017, c) 0.068, d) 0.102, e) 0.135, f) 0.169, and g) 0.330. The cell pathlength was 5 mm. aa base!ine 550 n m 500 450 400 350 146 Figure 7-5. Plots of % Change in CD at 360 nm as a Function of ATP/Av2 or ADP/Av2 Mole Ratio. The increase in CD was monitored at 360 nm and expressed as % change in CD with respect to the total change in CD. The amounts of nucleotide added were expressed in ATP/Av2 or ADP/Av2 molar r a tio . 1 E c o < x > oo + -> «3 Q O C •p- < u C 7 > c «3 O 20 — . 0.4 1 .2 2.0 2.8 3.6 ATP/ Av2 or ADP/AV2 Mole Ratio 4* 00 Figure 7-6. Plots of % Change in CD at 410 nm as Function of ATP/Av2 or ADP/Av2 Mole Ratio. The decrease in CD was monitored at 410 nm and expressed as % change in CD with respect to the total change in CD. The amounts of nucleotides added were normalized to per Av2 d imer. 100 o +-> Q O CD O ATP/AV2 or ADP/AV2 Mole Ratio VJl O Figure 7-7. Plots of % Change in CD at 470 nm as a Function of ATP/Av2 or ADP/Av2 Mole Ratio. The change in CD was monitored at 470 nm and expressed as % change in CD with respect to the total change in CD. The % change in CD was plotted against ATP/Av2 or ADP/Av2 molar ra tio s . The i n it ia l concentration of Av2 was 0.178 mM. MgADP or MgATP was added in I aliquots of a 0.20 m M solution of nucleotide. Either MgATP or MgADP were sequentially added to the protein cell sample. 151 C hange in C D a t 470 nm i 100 0.4 1 .2 2.0 2.8 3.6 ATP/AV2 or ADP/AV2 Mole Ratio U 1 IS) Figure 7-8. Effects of Av2 Specific A c tiv ity to the T itra tio n Curve at 360 nm. Curve A belongs to oxidized Av2 with SA of ^1400. Curve B belongs to oxidized Av2 with SA of ^2000. 153 ATP/AV2 M ole Ratio PGI % Change in C D at 360 n m r\3 -P * cn oo o o o o o o o o DO r o o ro oo REFERENCES 1. J.R. Postgate, Ed., "The Chemistry and Biochemistry of Nitrogen Fixation” , Plenum Press, New York, 1977. 2. W.E. Newton and W.H. Orme-Johnson, Eds., "Nitrogen Fixation", University Press, Baltimore, 1980. 3. C.E. McKenna, in "Molybdenum and Molybdenum Containing Enzymes", M.P. Coughlan, Ed., Pergamon Press, New York, 1980, ch. 14, pp. 439. 4. R.W.F. Hardy, Ed., "Dinitrogen F ixatio n ", Wiley, 1979. 5. L.E. Mortenson and R.N.F. Thorneley, Ann. Rev. Biochem. 48, 387 (1979). 6 . F. Bottom ley and R.C. 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Nyman, Eds., Washington State University Press, Pullman, WA, 1976, v o l. 1, p. 150. 109. A. Haystead and W.P.P. Stewart, Arch. M ikro b io l. 82, 1325 (1972) . 110. G.D. Watt and A. Burns, Biochem. 16. 264 (1977). 111. P.J. Stephens, C.E. McKenna, M.-C. McKenna, H.T. Nguyen, and D.J. Lowe, in "Iron and Proteins in Oxygen and Electron Transport", C. Ho, Ed., Elsevier, North-HolI and, 1980, in press. UMI Number: DP22681 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Di sser t at i on Pu bl i shi ng UMI DP22681 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. 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Creator Nguyen, Hop Trung (author)

Core Title Chiroptical spectroscopy of nitrogenase

School Graduate School

Degree Doctor of Philosophy

Degree Program Chemistry

Degree Conferral Date 1981-03

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, inorganic,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor McKenna, C.E. (committee chair), Petruska, John (committee member), Singer, Lawrence (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-65928

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