Study of cytochrome oxidase by the use of resonance Raman spectroscopy

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Content STUDY OF CYTOCHROME OXIDASE BY THE
USE OF RESONANCE RAMAN SPECTROSCOPY
by
Ronald Perreault
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
Master of Science
(Chemi s try )
September 1978
UMI Number: EP41665
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U N IVER SITY O F S O U TH E R N C A LIFO R N IA
THE GRADUATE SCHOOL
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LOS ANGELES, CALIFORNIA 9 0 0 0 7
This thesis, written by
Rojial.5 i..Perreau_l t ........................................................
under the direction of h}.^.....Thesis Committee,
and approved by a ll its members, has been pre
sented to and accepted by the Dean of The
Graduate School, in partial fulfillm ent of the
requirements fo r the degree of
M a_ster__of ..Science...................................
.............
Dean
Date Qe Z M v u 5 ^ .9 7 ?
e
Pf-55
THESIS COMMITTEE
if . fii
^ Chairman
Uiaurs.lJ
TABLE OF CONTENTS
Page
Lis t of Tab! es i i i
L is t o f F ig u r e s ............................................................................... iv
Abstract............................................................................................C . y i i
Introduction........................................................................................ 1
Technique ............................................................................................. 3
Biological Problem.......................................................................... 10
Application of Resonance Raman to Cytochrome
Oxidase........................................................................................ 21
Experi mental........................................................................................ 23
Experimental Results...................................................................... 32
Discussion............................................................................................. 75
Summary.................................................................................................. 88
References............................................................................................. 8$
i i
Table
1 .
2 .
3.
4.
LIST OF TABLES
Page
List of frequencies and polarization of
resonance Raman bands of hemeproteins
which are sensitive to spin and oxidation
state of metal .
List of strong vibrations
from 1 300 to 1650 cm- 1 .
List of strong vibrations
from 1300 to 1650 cm- 1 .
List of strong vibrations
from 1300 to 1650 cm"1.
List of vibrations in the
200 to 400 cm"1 .
8
in the region
81
in the region
82
in the region
83
region from
87
LIST OF FIGURES
Fi gure
1. Pictorial representation of electron
transport chain.
2. Chemical structure of hemes a and a^.
3. UV-VIS spectra of cytochrome oxidase.
4. Electron paramagnetic spectrum of oxidized
cytochrome oxidase.
5. Proposed structure of oxidized cytochrome
oxi dase.
6. Block diagram of the experimental set-up
for the accumulation of Raman spectra.
7. Resonance Raman spectrum of cytochrome
oxidase.
8. Resonance Raman spectrum of cytochrome
oxidase.
9. Resonance Raman spectrum of cytochrome
oxidase, no reducing equivalents added.
10. Electron paramagnetic spectrum of cytochrome
oxidase, no reducing equivalents added.
11. Resonance Raman spectrum of cytochrome
oxidase, .2 reducing equivalents added.
12. Resonance Raman spectrum of cytochrome
oxidase, .4 reducing equivalents added.
Pa ge
10
12
14
16
19
24
28
30
35
37
39
41
i v
Figure Page
13. Resonance Raman spectrum of cytochrome
oxidase, 1 reducing equivalent added. ' 43
14. Electron paramagnetic spectrum of
cytochrome oxidase, 1 reducing equivalent
added. 45
15. Resonance Raman spectrum of cytochrome
oxidase, 2 reducing equivalents added. 47
16. Electron paramagnetic spectrum of
cytochrome oxidase, 2 reducing equivalents
added. 49
17. Resonance Raman spectrum of cytochrome
oxidase, 4 reducing equivalents added. 51
18. Resonance Raman spectrum of cytochrome
oxidase, no added ligand. 53
19. Resonance Raman spectrum of cytochrome
oxidase, fluoride the added ligand. 55
20. Resonance Raman spectrum of cytochrome
oxidase, formate the added ligand. 57
21 . Resonance Raman spectrum of cytochrome
oxidase, cyanide the added ligand. 59
22. Resonance Raman spectrum of cytochrome
oxidase, sulfide the added ligand. 61
23. Resonance Raman spectrum of cytochrome
oxidase, reduced with no added ligand.
>
CO
VO
Fi gure Page
24. Resonance Raman spectrum of cytochrome
oxidase, reduced with fluoride the added
1i gand. 65
25. Resonance Raman spectrum of cytochrome
oxidase, reduced with formate the added
1i gand. 67
26. Resonance Raman spectrum of cytochrome
oxidase, reduced with cyanide the added
1i gand. 69
27. Resonance Raman spectrum of cytochrome
oxidase, reduced with sulfide the added
1i gand. 71
28. Resonance Raman spectrum of cytochrome
oxidase, reduced with azide the added
ligand. 73
29. Electron paramagnetic spectrum of
cytochrome oxidase with the addition of
one reducing equivalent. 76
30. Electron paramagnetic spectrum of
cytochrome oxidase with the addition of
one reducing equivalent. 78
31 . Proposed structure for the active site
of the resting cytochrome oxidase. 85
">yi
ABSTRACT
The mechanism of oxygen reduction by the metal lo-
enzyme, cytochrome oxidase, is s t i l l unknown. The
crystal structure of cytochrome oxidase has yet to be
determined. New indirect methods of studying of: the
active site of cytochrome oxidase have to be developed.
One of the new methods of studying metalloenzymes is
resonance Raman spectroscopy. The use of resonance
Raman spectroscopy to the study of the active site of
cytochrome oxidase was made. From these studies a
structure of the active site of cytochrome oxidase
will be proposed.
INTRODUCTION
In recent years a variety of spectroscopic methods
have been applied to the study of biological systems.
There are many d iffic u ltie s in applying spectroscopic
techniques to biological systems: sample preparation,
handling and interpretation of the data are a few. Many
types of samples are not readily available but have to
be prepared with procedures not always well established.
Proteins are sensitive and denature very easily under
non-physiological conditions. Data is frequently hard
to interpret because of the complexity of the molecules
being studied. The removal of the protein from its native
environment may cause subtle changes in the structure of
the protein which may not affect the function of the
protein but may a lte r the mechanism of the performed
function. The mechanism may be true in vitro but may be
to ta lly different in vivo. Experiments on biological
systems should be designed so that the data is easily
interpreted and the protein is as close to the physio
logical state as possible.
Even with the many d iffic u ltie s , significant use
of spectroscopic methods such as nuclear magnetic
resonance (NMR), u ltra v io le t and visible spectroscopy
(UV-VIS), circular dichroism (CD) and optical rotatory
dispersion (ORD) has been achieved. A summary of the
1
present uses of NMR, UV-VIS, CD, and ORD is. given:
NM R
(1) Verify the structure of proteins derived by
X-ray diffraction is valid in solution.
(2) Monitor transitions which occur in proteins
and polynucleotides on changes in pH and
temperature.
(3) Monitor the conformation of enzymes around the
active site.
(4) Study paramagnetic active sites in electron
transport proteins.
(5) Monitor the binding of the substrate to the
active site of an enzyme.
UV-VIS
(1) Determination of the concentration of a protein
in solution.
(2) Assay of a chemical reaction by the production
or disappearance of a chromophore.
(3) Monitor helix-coil transitions in DNA.
(4) Monitor protein association reactions.
CD-ORD
(1) Monitor the conformation of the enzyme on
binding of a substrate, change in temperature
and pH.
(2) Determination of the helical structure of
polynucleotides.
2
TECHNIQUE
A new spectroscopic technique which can be applied
to biological systems is resonance Raman spectroscopy.
A Raman spectrum is the spectrum of lig ht scattered
ine la stica lly from a sample. Upon scattering a photon
may occasionally lose a quantum of energy corresponding
to a molecular vibrational transition of the sample.
The Raman spectrum contains peaks of scattered light
whose frequency shifts correspond to the various vibra
tional frequencies of the scattering molecule. When,
as is usually the case in normal Raman spectroscopy, the
sample is transparent to the incident lig h t, energy
transfer from the photons a'rje i neffi ci ent and the Raman
spectrum is weak. High molecular concentrations (approxi-
mately .1 M or greater) are required. Moreover, there is
re la tiv e ly l i t t l e selectivity among the various vibrational
modes of the sample which for the complex molecules of
biology may be exceedingly numerous.
I f the wavelength of the inrcddent ligh t lies within
an electronic absorption of the sample, i.e . the incident
ligh t is close to resonance with an electronic transition,
then both sensitivity and selectivity are increased. The
vibrational modes which are expected to show enhancement
are the same ones which lend intensity to the electronic
spectrum; i.e . they are vibronically active modes. These
3
may be of two varieties: (A) modes which connect the
ground state to the excited state involved in resonance
through the Frank-Condon overlap or which change the
energy of the resonant excited state; and (B) modes which
mix the resonant electronic transition to another one
of higher energy. Type A vibrational modes are expected
to be to ta lly symmetric, while type B modes may have
any symmetry which is contained in the direct product of
the two electronic transition representations.
This theoretical framework serves admirably to explain
the resonance Raman scattering observed for heme proteins.
The visible and near u ltrav io let absorption spectra of
metal 1 oporphyrins are dominated by two allowed i t -*• i t *
electronic transitions. In molecular orbital terms these
originate in the two highest f ille d molecular orbitals of
A2u and A-ju symmetry under the molecular point group
and terminate in the lowest empty molecular orbital of Eg
symmetry. The two electronic transitions are both of Eu
symmetry and are subject to strong configuration in te r
action, with the result that the transition dipoles add
for the higher energy transition and largely cancel for
the lower one. The higher energy transition is assigned
5 -1 -1
to the very intense (e = 10 M cm ) absorption band,
called the Soret band, shown by all metal 1oporphyrins at
approximately 400 nm. The lower energy transition is
assigned to what is normally the f i r s t strong absorption
4
band, called the 3 band, which is found at approximately
550 nm and is an order of magnitude weaker than the Soret
band. The lower energy transition can borrow back some
of the intensity of the higher energy transitions through
appropriate vibrations. These are re lative ly high f r e
quency and produce a vibronic side band, called the a
band.
The heme protein Raman spectra contain a set of bands
which are in resonance with the Soret band. These are
of type A and are polarized to ta lly symmetric. Resonance
with the a band might also be expect to enhance type A
modes, but these have not been observed because their
intensity is too low, in consonance with the diminished
intensity of the alpha band. Rather, non-totally symmetric
bands are observed, whose intensity reach maxima at the
alpha peak, and also within the beta band, at positions
accurately predicted by the corresponding 0-1 frequency,
i.e . the frequency of the alpha peak plus the vibrational
frequency of the particular Raman band. These are type B
modes, which mix the two electronic transitions and give
rise to the beta band. Their allowed symmetries are
E = A, + A„„ + B, + B„ . The A, modes, however, have
u 1g 2g Ig 2g lg
been shown to be ineffective in vibronic mixing, and no
polarized Raman bands are found to be in resonance with
the alpha and beta bands. The B ^ and B^g modes give rise
to depolarized Raman bands, which are observed. The A£g
modes are inactive in normal Raman scattering, since they
are associated with antisymmetric scattering. Anti
symmetric scattering becomes allowed in the resonance
region, however, and gives to bands with inverse polariza
tion. The plane of polarization of the incident light
is rotated through 90° on scattering, and the polarization
ra tio , p, is in fin ity . The polarization ratio of a
particular vibration is defined by the expression
p = Ij^/111 where 1^ is the perpendicular intensity of the
vibration' '* and Ij| is the parallel intensity of the vibra
tion. The appearance of intense bands with inverse
polarization is the most remarkable characteristic of
heme protein resonance Raman spectra.
Some of the anomalous Raman bands have p-j = ~ within
experimental error; i.e . no scattering intensity is
observable in parallel polarization. But others have
appreciable intensity in parallel polarization and
3/4 < P -j < 00• W e characterize these as anomalously
polarized. I f the molecule has symmetry, then
modes should have p-j < °°. There are two possible explana
tions for a significant parallel component of anomalously
polarized modes. The effective symmetry of the molecule
may be less than , in which case the formerly &2g
modes may acquire symmetric as well as antisymmetric
components. Alternatively there may be accidental
degeneracies between A2g modes and modes of other
6
symmetries, which are polarized (A-jg)-or depolarized (Blg
or B2 g)> giving an overall depolarization ratio which is
anomal otis .
Hemeprotein vibrationals are assigned by their
polarization and their frequency. Spiro [1,2 ,3 ] has found
that certain Raman bands are sensitive to the spin and
oxidation state of the metal, iron, in the hemeprotein.
Table 1 lis ts the frequencies and polarization of the
bands which are sensitive to spin and oxidation state of
the metal. The sensitivity of Raman bands to spin state
of the metal is caused by changes in the geometry of the
iron porphyrin [4]. In high spin iron porphyrins, the
iron is always out of the porphyrin plane. Upon going low
spin, the iron moves into the porphyrinplane. The force
constants of the porphyrin bonds when the iron is in the
place of the porphyrin are different from those when the
iron is out of the plane. The change in force constants
results in shifting of the porphyrin vibrations. The
sensitivity of Raman bands to the oxidation state of the
metal can be understood in terms of changes in t t back
donation of electrons from the central iron atom to the
porphyrin ring [5]. The iron d orbitals are more
extended for F e (II) than for F e ( I I I ) , because of the
lower effective charge, and overlap with porphyrin t t *
orbitals is greater. Populating the t t * orbitals results
in weakening the porphyrin ring bonds and lowering their
TABLE 1
List of frequencies and polarization of resonance Raman bands of
hemeproteins which are sensitive to spin and oxidation state of metal.
Spin
State
Oxi dati on
State Molecule
Oxidation state
markers
A(p) B(dp)
Spin state
markers
C(ap) D(p)
Oxidation and spin
state markers
E(p) F{dp)
1 s F e (III) Ferricyt c 1374 1562
1582 1582 1502 1636
Is F e (III) CNMHbb 1374 1564 1588 1583 1508 1642
hs F e (III) FMHbb 1373 1565
1555 1565 1482 1608
hs Fe(II) Deoxy Hbb 1358 1546
1552 1565 1473 1607
Is Fe(II) Ferrocyt c 1362 1548
1584 1594 1493 1620
Vs
- ?
Oxy Hbb 1377 1564
1586 1582 1506 1540
Low spin
o o
, Is; high spin, hs. bHb, hemoglobin ; MHb, methemoglobi n.
force constants. The observed lowering of vibrational
frequencies associated with the stretching of porphyrin
ring bonds can be understood on this basis.
BIOLOGICAL PROBLEM
The cells in the body need to function. The food we
eat cannot be used directly to perform many cell functions.
The food is u tilized as a substrate in a number of exo
thermic reactions. The energy given off in these reactions
is used to synthesize ATP which can be used to perform cell
functions. One of the sites where ATP is produced is the
inner membrane of the mitochondria. The site in the inner
membrane of the mitochondria is called the electron
transport system. The electron transport system is made
up of four complexes and three sites where ATP can be
produced. A pictorial representation of electron transport
chain is shown below
, ADP
+ ATP
P.
NAD
ADP
ATP + ATP
X X
zc
Complex I CoQ Complex I I I Cyt c . Complex jIV
NADH + H
FADH, FAD
Complex II
.pyruvate
malate
- i s o c i t r a t e
^ a - ketog1utarate
Figure 1
Pictorial representation of electron transport chain
10
The interests in this laboratory is Complex IV, also
called cytochrome oxidase. Cytochrome oxidase catalyzes
the reduction of oxygen to water. The reduction of oxygen
to water is an exothermic process; the energy released is
used to synthesize ATP. Cytochrome oxidase is a metallo-
enzyme of molecular weight of 150,000 to 200,000. The
enzyme's minimal functional unit contains two moles of
heme, designated a and a3> and two moles of copper,
designated Cug and Cu^. The two hemes are different even
though their chemical structures are the same [6]. Figure
2 shows the structure of hemes a and a^. Figures 3 and .4
are the UV-VIS and electron paramagnetic resonance (EPR)
spectra of the resting, oxidized enzyme. The differenees
in the hemes arise from differences in spin states and
reactivity to exogenous ligands. Heme a is unable to
bind exogenous ligands while heme a^ is able to bind
sulfide, cyanide, formate, fluoride, carbon monoxide,
n itric oxide, and oxygen. The magnetic circular dichroism
(MCD) spectrum of oxidized cytochrome oxidase [7] detects
a high and a low spin heme. The MCD spectrum of the
reduced enzyme again detects a low and a high spin heme
signals. The low spin signal in both the oxidized and
reduced cytochrome oxidase is assigned to heme a and the
high spin signal to heme a3 -
The EPR spectrum of the resting enzyme exhibits an
ax ially symmetric resonance [8] close to g = 2 (similar to
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ I I
Figure 2-. Chemical structure of hemes a and a3
1 2
H O
HO H O O H H O
("HO) HO H O H O H O
HO H O H O
Figure 3 UV-VIS spectra of cytochrome oxidase
(Ref. 27).
14
solid line = oxidized
broken line = reduced
o
350 450 550 650
Wavelength (my)
cn
Figure & EPR spectrum of oxidized cytochrome
oxidase.
16
o
to
C M
C D ,
CM
C O
O
09
< 1 1 '
cn
o
o
C 7 >
O
3
17
Type 1 copper proteins). A second species identified by
EPR is a low-spin heme present in the resting enzyme [9].
The EPR spectrum of p a rtia lly reduced cytochrome oxidase
exhibits a new high spin heme signal. There is, however,
+ 2
a major dilemma in that the intensity of both the Cu
+ 3
and Fe resonance observed in the resting enzyme only
+ 2 +3
accounts for .8 mole of Cu and 1.0 mole of Fe per
functional unit.
A second puzzling property of resting cytochrome
oxidase is its poor reactivity with reagents recognized
as effective fe rric heme ligands. For example, while the
reaction of the oxidase with sodium azide is rapid, the
changes in the visible spectrum are extremely small [7]
and make i t quite unlikely that the reaction of azide is
with heme iron. This is in contrast to the ready reaction
of p a rtia lly reduced oxidase with azide which results
in the conversion of a high-spin EPR signal to a low-spin
one and demonstrates that the reaction is indeed with a
heme iron under these conditions. Cyanide [10] and
sulfide [1.1] behave sim ilarly and i t seems credible that
the heme sites in the resting oxidase are blocked, but,
upon reduction, a structural change occurs which exposes
the liganding site to exogenous reagents.
The peculiar properties of the resting enzyme could
be answered by proposing the following structure:
18
Figure 5. Proposed structure of oxi di zed cytochrome oxi dase
,x
HISTIDINE
HEME
Heme a3 is antiferromagnetically couple to Cub through
the ligand X giving no EPR signals fromheme a^ and Cu^.
What is meant by a n ti- ferromagnetic coupling is that the
single unpaired electron of Cub couples with one of the
five unpaired electrons of the heme a^, resulting in
no detectable EPR signal from Cu^. Heme a3 has now four
unpaired electrons and is EPR undetectable. The visible
EPR signals are assigned to Cu, and heme a.
a
In the proposed structure for the resting enzyme,
heme a^ is six coordinate and would not accept another
ligand into its coordination sphere. The partial or fu ll
reduction of the enzyme causes a conformation change in the
enzyme [12], which moves the ligand X from heme a3
breaking the coupling between Cub and heme a^. Heme a 3 's
sixth-coordination site is no longer blocked and accepts
typically good heme ligands, sulfide, cyanide, and azide.
The mechanism of oxygen reduction by cytochrome
oxidase is unknown. The proposed structure supports the
idea of a two metal binding site for oxygen [13,14].
T9
Oxygen upon binding to the active enzyme would bridge heme
a3 and Cub- The in it ia l step in the reaction would be a
two electron transfer to the oxygen from heme a3 and Cub
_ 2
to form the peroxide, 09 , intermediate.
20
APPLICATION OF RESONANCE RAMAN TO CYTOCHROME OXIDASE
The resonance Raman spectra of many hemeproteins have
been studied [1-3,15-17]. I t has been found that the
resonance Raman spectrum of hemeproteins is sensitive to
the electronic state of the metal and the geometry of
the iron porphyrin. This sensitivity has led to the
elucidation of the active site of hemoglobin. The appli
cation of resonance Raman to the study of the active site
of cytochrome oxidase is complicated by having two hemes
in the functional unit. Hemes a and a3 contribute
equally to the electronic absorption at 423 nm of the
resting enzyme so both heme vibrations are selectively
enhanced. The Raman vibrations from heme a3, the active
site of the enzyme, could be determined by use of p o lari
zation measurements, inhibitor compounds and redox t i t r a
tions.
Heme a3 is high spin both the oxidized and reduced
form of the enzyme while heme a is low spin. Heme a3 is
able to bind exogeneous ligands, cyanide, sulfide, azide,
fluoride, and formate, while heme a is not. Strong
ligands such as cyanide, sulfide and azide affect a spin
transition in heme a3- In the spectra of cytochrome
oxidase with and without a strong ligand, any shifted
vibrations are assigned to heme a3 - The redox titra tio n s
performed by Beinert et a l . [18] indicate that the order
21
of reduction of the metal centers in cytochrome oxidase is
heme a, Cu^, heme a^ and Cua - The addition of two equi
valents of electrons to cytochrome oxidase reduces heme a
and Cub; heme a3 and Cub are s t i l l oxidized. In the
spectra of cytochrome oxidase with and without two equi
valents of electrons, any shifted vibrations are assigned
to heme a.
The resonance Raman spectrum of cytochrome oxidase
has been studied by Nafie et a l . [19] and Salmeen et a l .
[20]. The interesting feature of these spectra is in the
region between 1500 and 1 700 cm"'' where four strong
vibrations are observed. I f the spectrum of high and
low spin hemeproteins are added four strong vibrations
are found in this region. In the spectrum of cytochrome
c and hemeglobin, the region between 1500 and 1700 cm~^
was most sensitive to the spin state of the metal.
Because of this information and the differences of spin
states between hemes a and a^, i t may be possible to
assign two of the vibrations to heme a and two to heme a^.
Salmeen and Nafie did not try to assign the four vibrations.
The use of inhibitor compounds and redox titra tio n s may
lead to the assignment of these vibrations and the better
understanding of the active site of cytochrome oxidase.
22
EXPERIMENTAL
A block diagram of the experimental set-up for the
accumulation of Raman spectra is illu stra ted in Figure,6.
The v e rtica lly polarized lig ht from the laser was passed
through a half wave plate to obtain horizontally polarized
lig ht. After reflection from the mirror C, the light was
then focused onto the sample. The scattered light was
focused on the monochromator s l i t by a collecting lens.
The signal from the photomultiplier was then sent through
a preamplifier and was detected by a photon counter
followed by a rate metdr connected to a recorder. Raman
spectra were obtained by scanning the monochromator at
speeds of 20 cm~^ per minute with 10 second time constants
and a resolution of 5 cm”^. The lig h t sources were a
Coherent Radiation model CR-3 Argon Ion laser and Liconix
model 411 O H Helium Cadium laser. The laser power was 10
to 100 m illiw atts. The monochromator used was a Spex
1401 double spectrometer equipped with an ITT FW 130
electronically cooled photomultiplier tube. A SSR model
1105 photon counter and a RCA 1120 preamplifier were used
for photon counting. The power supplied to the photo
m ultiplier tube was about 1500 volts and a typical signal
was about 1000 counts with slitwidths set at 200 microns
which gave a spectral resolution of about 5 cm~^.
As mentioned, the resonance Raman spectrum of
23
Fi gure 6 A block diagram of the experimental
set-up for the accumulation of Raman
spectra.
24
A = ARGON ION LASER
B = HALFWAVE PLATE
C = MIRROR
D = LENS
E = LO W TEMPERATURE RAMAN CELL
F = MONOCHROMATOR
G = PREAMPLIFIER
H = PHOTON COUNTER
I = RECORDER
cytochrome oxidase has been taken. Completely analogous
conditions were tried but good quality spectra could not
be obtained. The reasons for failure were:
(1) Background was large and weak Raman signals
could not be detected.
(2) The protein was heated by the light source
causing the decompos i tion of the sample.
The large background was caused by the method of
purification of the enzyme, cytochrome oxidase. Cyto
chrome oxidase is d if f i c u l t to purify because i t is a
membrane bound protein. The methods of preparation
followed were that of Yonetani [21] and Sun [22]. The
method of preparation ^sj. to breakdown the membrane wall
by sonification and solubilize the membrane bound proteins
by use of a detergent. In the preparation of Yonetani
the detergent used was deoxycholate while in the Sun
preparation Triton X-100. The proteins are selectively
precipitated by the use of a s a lt, in the Yonetani prepara
tion ammonium sulfate and in the Sun preparation potassium
chloride. The background of the Sun preparation was a
factor of three less than the Yonetani preparation. The
added background in the Yonetani preparation was from the
deoxycholate.
The usual Raman cell for hemeproteins is the melting
point capillary. The localized heating of the melting
point capillary by the ligh t source caused the decomposition
26
of the enzyme. The known methods of sample holding to
prevent heating are the spinning cell and the flow through
capillary. Both techniques proved to be unsuccessful
because they increased the background and Raman could not
be detected. A low temperature cell to prevent heating
was built but spectrum could not be obtained.
The intensity of the lig h t source affects the amount
of heating of the sample, i f the laser power was less than
10 milliwatts no noticeable sample decomposition was
detected. The in it ia l studies used an Argon Ion laser
with an exciting line at 457.9 nm. The lin e a t 457.9 nm
is about 30 nm away from the electronic absorption
maximum of the oxidized enzyme and 10 nm from the maximum
of the reduced enzyme. The intensity of Raman lines [23]
is inversely proportional to the frequency difference
between the incident ligh t and the maximum of the elec
tronic transition. By the use of a 457.9 nm excitation,
maximum laser power had to be used to get poor quality
spectra, Figure ‘7. At maximum power (100 m illiw atts)
much decomposition of the sample was detected. I f the
ligh t source was closer in frequency to the electronic
absorption of the enzyme, less laser power could be used
to get good quality spectra. The light source used was a
He!ium-Cadiurn laser with an exciting line at 441.6 nm.
Using the 441.6 lig h t source and minimal power (10 m i l l i
watts) excellent quality spectra, Figure 8, were obtained
with no visible sign of decomposition._____________________
Figure 7. Resonance Raman spectrum of cytochrome
oxi dase.
Spectral conditions: excitation line =
457.9 cm; spectral s l i t width = 5 cm"^,
scan speed = 20 cm" Vmi n, time constant =
10 sec.
28
-H -------------------1 -------------------1 ------------------ 1 -------------------1 ------------------- 1 ------------------- 1 ------------------ 1 ------------------ 1 ------------------- 1 -------------------1 -------------------1 ----
300 425 550 675 800 925 1050 1175 1300 1425 1550 1675
ro
< o
Figure 8. Resonance Raman spectrum of cytochrome
oxi dase.
Spectral conditions: excitation line =
441.6 nm, spectral s l i t width = 5 cm- ^ ,
scan speed = 20 cm'Vmin, time constant =
10 sec.
30
100 . 300 500 700 900 1100 1300 1500 1700
EXPERIMENTAL RESULTS
The resonance Raman spectra of cytochrome oxidase
are obtained at various points during the redox titra tio n .
The redox titra tio n s were monitored by EPR spectroscopy
and the resulting spectra are shown.
Figure r9, resonance Raman spectrum of cytochrome
oxidase, no reducing equivalents added.
Figure TO , electron paramagnetic spectrum of cyto
chrome oxidase, no reducing equivalents added.
Figure Ifl," resonance Raman spectrum of cytochrome
oxidase, .2 reducing equivalents added.
Figure 12, resonance Raman spectrum of cytochrome
oxidase, .4 reducing equivalents added.
Figure 13, resonance Raman spectrum of cytochrome
oxidase, 1 reducing equivalent added.
Figure 14, electron paramagnetic spectrum of cyto
chrome oxidase, .1 reducing equivalent added.
Figure 15,- resonance Raman spectrum of cytochrome
oxidase, 2 reducing equivalents added.
Figure 16, electron paramagnetic spectrum of cyto
chrome oxidase, 2 reducing"equivalents added.
Figure 1 *7, resonance Raman spectrum of cytochrome
oxidase, 4 reducing equivalents added.
The resonance Raman spectra of cytochrome oxidase
are obtained in differen t oxidation and spin states. The
32
enzyme is fu lly reduced by the addition of excess ascorbate
and the spin state of heme a3 is manipulated by the addi
tion of exogeneous ligands.
I
Figure A 8", resonance Raman spectrum of cytochrome
oxidase, heme a F e ( III) low spin, heme a^ F e ( I II ) high
spin, no added ligand.
i t
Figure 19-, resonance Raman spectrum of cytochrome
oxidase, heme a F e ( III) low spin, heme a3 F e ( III) high
spin, fluoride the added 1igand.
Figure 20, resonance Raman spectrum of cytochrome
oxidase, heme a F e ( III ) low spin, heme a3 F e ( III) high
spin, formate the added ligand.
Figure 21, resonance Raman spectrum of cytochrome
oxidase, heme a F e ( III) low spin, heme a3 F e ( III) low
spin, cyanide the added ligand.
Figure 22, resonance Raman spectrum of cytochrome
oxidase, heme a F e ( III) low spin, heme a3 F e ( III) low
spin, sulfide the added ligand.
Figure 23, resonance Raman spectrum of cytochrome
oxidase, heme a F e ( II) , low spin, heme a3 F e (II) high
spin, no added ligand.
Figure 24, resonance Raman spectrum of cytochrome
oxidase, heme a F e (II) low spin, heme a3 F e (II) high
spin, fluoride the added ligand.
Figure 25, resonance Raman spectrum of cytochrome
oxidase, heme a F e (II) low spin, heme a3 F e (II) high
_________________________________________________________________ 3 _ 3 _
spin, formate the added ligand.
Figure -26, resonance Raman spectrum
oxidase, heme a F e (II) low spin, heme a3
spin, cyanide the added ligand.
Figure 2-7, resonance Raman spectrum
oxidase, heme a F e (II) low spin, heme a3
spin, sulfide -the added ligand.
Figure 28, resonance Raman spectrum
oxidase, heme a F e (II) low spin, heme a3
spin, azide the added ligand.
of cytochrome
F e (II) low
of cytochrome
F e (II) low
of cytochrome
F e (II) low
Figure 9. Resonance Raman spectrum of cytochrome
oxidase, no reducing equivalents added.
Spectral conditions: excitation line =
441.6 nm, spectral s l i t width = 5 cm~^,
scan speed = 20 cm”Vm in, time constant =
10 sec. Concentration of solution:
_ 5
cytochrome oxidase = 3.1x10 ,
Triton X-100 = 5%.
35
1100 1300 1500 1700
Figure 10. Electron paramagnetic spectrum of
cytochrome oxidase, no reducing
equivalents added.
Spectral conditions: scan range =
3
4x10 , time constant = .1 sec,
2
receiver gain = 6.2x10 , microwave
power = 15 mW , fie ld set = 2900 gauss,
scan time = 2 minutes, modulation
frequency = 100 K, temperature = 10°K.
Concentration of solution : cytochrome
oxidase = 3.1x10"^, Triton X-100 = 5%.
37
o
in
cn
o
C X I
C D
CO
O
CO
o
o
o
o
38
Figure 11. Resonance Raman spectrum of cytochrome
oxidase, .2 reducing equivalents added.
Spectral conditions: excitation line =
441.6 nm, spectral s l i t width = 4 cm- ^,
scan speed = 20 cm"^, time constant =
10 sec. Concentration of solution:
_ 5
cytochrome oxidase = 2.48x10 mole/1,
Triton X-100 = 5%, cytochrome c =
2.48x10"6 mole/1, EDTA = 4.0xl0~7 mole/1,
ascorbate = 4.98x10"^.
39
A
■ 200 400 600 800 1200 1400 1600
o
Figure 12. Resonance Raman spectrum of cytochrome
oxidase, .4 reducing equivalents added.
Spectral conditions: excitation line =
441.6, spectral s l i t width = 4 cm”^,
scan speed = 20 cm"^, time constant =
10 sec. Concentration of solution:
_ 5
Cytochrome oxidase = 2.48x10 ,
Triton X-100 = 5%, cytochrome c =
2.48x10-6 mole/1, EDTA = 8.0xl0"7 mole/1,
ascorbate = 8.96x10"® mole/1.
41
1600 1800 400 800 1400 200 600 1000 1200
Figure 13. Resonance Raman spectrum of cytochrome
oxidase, 1 reducing equivalent added.
Spectral conditions: excitation line =
441.6, spectral s l i t width = 4 cm~^ ,
scan speed = 20 cm~Vmin, time constant =
10 sec. Concentration of solution:
_ 5
cytochrome oxidase = 2.48x10 mole/1,
Triton X-100 = 5%, cytochrome c =
2.48x10"5 mole/1, EDTA = 2.0xl0 " 6 mole/1,
ascorbate = 2.49xl0- 5 mole/1.
43
400 600 800 1400 1200 1600 1800
Figure 14. Electron paramagnetic spectrum of
cytochrome oxidase, 1 reducing
equivalent added.
Spectral conditions: scan range =
3
4.0x10 , time constant = .1 sec,
2
receiver gain = 6 . 2 x1 0 , microwave
power = 15 m W , fie ld set = 2900 gauss,
scan time = 2 minutes, modulation
frequency = 100 K, temperature = 10°K.
Concentration of solution: cytochrome
oxidase = 2.48x10“^ mole/1, Triton X-100 =
_ 5
5%, cytochrome c = 2.48x10 mole/1,
EDTA = 2.0x10”6, ascorbate = 2.49xl0" 5
mol e / 1 .
45
o
L O
C O
o
C M
II
C D
CM
C T >
C O
O
C O
c n
o
o
c n
o
o
46
I
Figure 15. Resonance Raman spectrum of cytochrome
oxidase, 2 reducing equivalents added.
Spectral conditions: excitation line =
441.6 nm, spectral s l i t width = 4 cm- ^,
scan speed = 20 cm~Vmin, time constant =
10 seconds. Concentration of solution:
_ 5
cytochrome oxidase = 2.48x10 mole/1,
Triton X-100 = 5%, cytochrome c =
2.48x1O-6 mole/1, EDTA = 4.0xl0 " 6 mole/1,
ascorbate = 4.98xl0~5 mole/1.
47
400 200 800 1 ' 4 0 0 1600 600 1000
( CJT1-1 )
1200 1800
Figure 16. Electron paramagnetic spectrum of
cytochrome oxidase, 2 reducing
equivalents added.
Spectral conditions: scan range =
3
4.0x10 , time constant = .1 sec,
2
receiver gain = 6 . 2 x1 0 , microwave
power = 15 m W , fie ld set = 2900 gauss,
scan time = 2 minutes, modulation
frequency = 100 K, temperature = 10°K.
Concentration of solution: cytochrome
oxidase = 2.48xl0~5 mole/1, Triton X-100 =
5%, cytochrome c = 2.48x10"^ mole/1,
EDTA = 2.0x10"^ mole/1, ascorbate =
4.98x10"5 mole/1 .
49
o
ID
< £ >
O
O J
C D
CM
C D
C O
o
o
o
C D
O
C D
50
Figure 17. Resonance Raman spectrum of cytochrome
oxidase, 4 reducing equivalents added.
Spectral conditions: excitation line =
441.6 nm, spectral s l i t width = 5 cm“^,
scan speed = 20 cm- V m in, time constant =
10 seconds. Concentration of solution:
_ 5
cytochrome oxidase = 6 . 2 x1 0 mole/1 ,
Triton X-100 = 5%, cytochrome c =
8.3x10"^ mole/1, ascorbate = 3.3x10"^
mole/1, EDTA = l.OxlO” 3 mole/1.
51
\ I 1 I » » \
150 350 550 750 950 1150 1350 1550 1750
________________ . __________________v (cm~^ )___________________________________________
Figure 18. Resonance Raman spectrum of cytochrome
oxidase, no added ligand.
Spectral conditions: excitation line =
441.6 nm, spectral s l i t width = 5 cm- ^,
scan speed = 2 0 cnT^/min, time constant =
10 seconds. Concentration of solution:
_ 5
cytochrome oxidase = 6 . 2 x1 0 mole/1 ,
Triton X-100 = 5%.
53
100 300 500 700 900 1100 1300 1500 1700
v (cm~^ )
cn
-P *
Figure 19. Resonance Raman spectrum of cytochrome
oxidase, fluoride the added ligand.
Spectral conditions; excitation line =
441.6 nm, spectral s l i t width = 5 cm"^,
scan speed = 20 cm~Vnnn, time constant =
10 seconds. Concentration of solution;
_ 5
cytochrome oxdiase = 6 . 2 x1 0 mole/1 ,
Triton X-100 = 5%, sodium fluoride =
.1 mole/ 1 .
C l
250 450 650 850 1050 1250 1450 1650
ui
cn
Figure 20. Resonance Raman spectrum of cytochrome
oxidase, formate the added ligand.
Spectral conditions: excitation line =
441.6 nm, spectral s l i t width = 5 cm”^,
scan speed = 2 0 cm- Vmin, time constant =
10 seconds. Concentration of solution:
_ 5
cytochrome oxidase = 6 . 2 x1 0 mole/1 ,
Triton X-100 = 5%, sodium formate =
2 . 2 x1 0 " 2 mole/1 .
57
T O O 300 500 700 900
v (cm” ^)
1100 1300 1500 1700
cn
00
fo ft vi
Figure 21. Resonance Raman spectrum of cytochrome
oxidase, cyanide the added ligand.
Spectral conditions: excitation line =
441.6 nm, spectral s l i t width = 5 cm‘ \
scan speed = 2 0 cnT^/min, time constant =
10 seconds. Concentration of solution:
_ 5
cytochrome oxidase = 6 . 2 x1 0 mole/1 ,
Triton X-100 = 5%, potassium cyanide =
3.05xl0"2 mol e /1 .
59
450 250 850 ' 1250 1450 650 1650
0 " >
o
Figure 22. Resonance Raman spectrum of cytochrome
oxidase, sulfide the added ligand.
Spectral conditions: excitation line =
441.6 nm, spectral s l i t width = 5 cm~\
scan speed = 20 cm~Vnnn, time constant =
10 seconds. Concentration of solution:
_ 5
cytochrome oxidase = 6 . 2 x1 0 mole/1 ,
Triton X-100 = 5%, sodium sulfide =
2 . 0 x1 0 ”^ mol e / 1 .
61
30 Q 500 700 900 1100
c r >
ro
Figure 23. Resonance Raman spectrum of cytochrome
oxidase, reduced with no added ligand.
Spectral conditions: excitation line =
441.6 nm, spectral s l i t width = 5 cm "\
scan speed = 20 cm- V m in, time constant =
10 seconds. Concentration of solution:
_ 5
cytochrome oxidase = 6 . 2 x1 0 mole/1 ,
Triton X-100 = 5%, cytochrome c =
8.3x10"^ mole/1, ascorbate = 3.3x10“^
m o 1 e/ 1 .
63
450 2 50 850 1050 12 50 1450 1650
v ( cm-1 )
Figure 24. Resonance Raman spectrum of cytochrome
oxidase, reduced with fluoride the
added ligand.
Spectral conditions: excitation line =
441.6 nm, spectral s l i t width = 5 cm~\
scan speed = 20 cm- V m in, time constant =
10 seconds. Concentration of solution:
_ 5
cytochrome oxidase = 6 . 2 x1 0 mole/1 ,
Triton X-100 = 5%, cytochrome c =
5.0x10”^ mole/1, ascorbate = 2.5x10"^
mole/1, sodium fluoride = 1.25x10"^
mole/ 1 .
E (U
in c
* i —
CD Q -
Q .
Q .
__)--------------------------- ( ----------------------------1 ----------------------------1 ---------------------------- 1 ----------------------------1 ---------------------------- 1 ----------------------------j----------------------------1 -
100 300 500 700 900 1100 1300 1500 1700
C l
o
Figure 25. Resonance Raman spectrum of cytochrome
oxidase, reduced with formate the added
ligand.
Spectral conditions: excitation line =
441.6 nm, spectral s l i t width = 5 cm~^,
scan speed = 20 cm“ V m in, time constant =
10 second. Concentration of solution:
_ 5 •
cytochrome oxidase = 6.2x10 mole/1,
Triton X-100 = 5%, cytochrome c =
5.0xl0 “ 6 mole/1, ascorbate = 2.5xl0 ~ 2
- 2
mole/1, sodium formate = 2.75x10 mole/1.
67
e < u
to c
fO *r
Q .
to
Q-
to
CL
1 0 0 300 500 700 900 1100 1300 1 500 1700
v (cm
c r>
o o
Figure 26. Resonance Raman spectrum of cytochrome
oxidase, reduced with cyanide the added
ligand.
Spectral conditions: excitation line =
441.6 nm, spectral s l i t width = 5 cm- 1 ,
scan speed = 2 0 cnT^/min, time constant =
10 seconds. Concentration of solution:
_ 5
cytochrome oxidase = 6 . 2 x1 0 mole/1 ,
Triton X-100 = 5%, cytochrome c =
5x0x10"^ mole/1, ascorbate = 2.5x10"^
_ ?
mole/1, potassium cyanide = 4.3x10
mol e/ 1 .
69
1700 1500 1300 1100 900 700 500 300 100
o
Figure 27. Resonance Raman spectrum of cytochrome
oxidase, reduced with sulfide the added
1 i gand.
Spectral conditions: excitation line =
441.6 nm, spectral s l i t width = 5 cm"^,
scan speed = 2 0 cm- 1/mi n, time constant =
10 seconds. Concentration of solution:
_ 5
cytochrome oxidase = 6 . 2 x1 0 mole/ 1 ,
Triton X-100 = 5%, cytochrome c =
5.0x10“^ mole/1, ascorbate = 2.5x10”^
_ ?
mole/1, sodium sulfide = 2.5x10 mole/1.
71
C l
Q . i/J
C L
T O O 300 500 700 900 1100 1300 1 500 1700
v (cm
Figure 28. Resonance Raman spectrum of cytochrome
oxidase, reduced with azide the added
1 i gand.
Spectral conditions: excitation line =
441.6 nm, spectral s l i t width = 5 cm~\
scan speed = 2 0 cm- Vmin, time constant =
10 seconds. Concentration of solution:
_ 5
cytochrome oxidase = 6 . 2 x1 0 mole/1 ,
Triton X-100 = 5%, cytochrome c =
5.0xl0 " 6 mole/1, ascorbate = 2.5xl0 - 2
- 2
mole/1, sodium azide = 2.65x10 mole/1.
73
0)
c
fd
s < u
I/) c
fO *r-
C l
Q .
100 300 500 700 900 1100 1300 1 500 1700
(cm~^ )
DISCUSSION
As is the usual case, the data for biological
systems is hard to interpret and is not always consistent
with what previous workers have done. The differences
might arise from d ifferen t methods of preparation or
sample handling. The protein has been removed from the
native environment and small changes in suspending media,
i . e . , pH, buffer, detergent, might cause the differences
in results. The results of the EPR monitored redox t i t r a
tions presented here d iffe r from those performed by
Vanngard [24], Beinert [25], and Hartzell [26]. They
found a new high spin Fe signal that resulted from the
addition of one reducing equivalent, Figure 29. They
subsequently assigned this signal to heme a3 - Upon
addition of one equivalent of electrons in the experiments
performed here, no new high spin Fe signal was detected,
Figure 36. The exact assignment of which component,
heme a or heme a3> is reduced f i r s t is not necessary for
the study. The important feature is that one heme is
reduced while the other is oxi'dized. From the EPR spectra
presented here and other data from this laboratory, heme
a3 is reduced before heme a.
The addition of one equivalent of electrons reduces
heme a3 but maintains heme a in the oxidized form. Upon
addition of one equivalent of electrons, vibrations which
75
Figure 29. Electron paramagnetic spectrum of
cytochrome oxidase with the addition
of one reducing equivalent (reference
25) .
c n
C M
c n
O
o
C O
II
C T >
77
Figure 30 Electron paramagnetic spectrum of
cytochrome oxidase with the addition
of one reducing equivalent.
78
o
LO
cn
O
C X I
cn
c \ j
CO
o
CO
CD
O
O
c n
o
o
79
are sensitive to the oxidation state of the metal should
sh ift to lower vibrational frequency. The vibrations
which s h ift are assigned to heme a^. Table 2 lis ts the
strong vibrations in the Raman spectrum of cytochrome
oxidase in the oxidation sensitive region at different
amounts of added electron equivalents. The vibration
at 1618 crn- ^ in the oxidized enzyme shifts to 1601 cm- " *
upon addition of 1 equivalent of electrons. The vibration
at 1618 cm~^ is assigned to heme a3 -
- 1
The vibration at 1618 cm is sensitive to the oxi
dation state of the Fe in heme a^. This vibration should
also be sensitive to the spin state of the Fe in heme a^.
Tables 3 and 4 lis ts the strong vibrations in the Raman
spectrum of cytochrome oxidase in the spin sensitive
region at d ifferen t spin states of heme a^. A change in
spin states of the heme a3 does not effect the 1618 cm- ^
vibration or any other vibrations in the spin sensitive
region of the Raman spectrum. In other heme proteins
[1-3,15-17] a change of spin state causes considerable
shifts in vibrations in the region from 1550 to 1770 crn"^.
The shifts are caused by changes in the geometry of the
iron porphyrin when the spin state changes. In five
coordinate high-spin porphyrins the iron is always out of
the porphyrin plane; heme a3 is assumed to be of this type.
The addition of a ligand such as cyanide, drives the iron
low spin, moving the iron into the porphyrin plane. The
80
TABLE 2
List of strong vibrations in the region from 1300 to 1650 cm- ^.
0 equivalents .2 equivalents .4 equivalents 1 equivalent 2 equivalents 4 equivalents
of electrons of electrons of electron of electrons of electrons of electrons
1362
— 1
cm 1362
rm" 1
cm 1361
rm- 1
cm 1361 cm 1363 cm~^ 1361 cm
1578
- 1
cm 1570
- 1
cm 1573
- 1
cm 1573
- 1
cm
1593 cm 1590 1590
- 1
cm 1590
- 1
cm 1585
- 1
cm 1590
- 1
cm
1617
- 1
cm 1618
rn, ’ 1
cm 1605 cm 1601
rnT1
cm 1620
~ 1
cm 1620 cm~^
1630
- 1
cm 1630
- 1
cm 1630
- 1
cm 1630
- 1
cm 1630
- 1
cm 1630
- 1
cm
0 0
TABLE 3
List of strong vibrations in the region from 1300 to 1650 cm"1.
heme a F e (III)
low spin
heme a F e (III)
low spin
heme a Fe( 111)
low spin
heme a F e (III)
low spin
heme F e (III)
high spin
heme a3 Fe( 111)
high spin
fluoride added
heme a3 F e (III)
low Spin
cyanide added
heme a3 F e (III)
low spin
sulfide added
1362 cm " 1 1362 cm' 1 1362 cm 1364 cm"
1578 cm" 1 1578 cm" 1 1576 cm 1570 cm"
1593 cm" 1 1592 cm' 1 1595
- 1
cm 1592 cm
1617 cm " 1 1617 cm" 1 1615 cm"
1630 cm" 1 1630 cm' 1 1628
- 1
cm 1630 cm"
0 0
ro
Table 4
List of strong vibrations in the region from 1300 to 1650 c m ' 1
h em e a Fe(II)
low spin
h em e Fe(II)
high spin
h em e a Fe(II)
low spin
h em e a3 Fe(I I )
high spin
fluoride added
h em e a Fe(II)
low spin
h em e a3 Fe(II)
high spin
formate added
h e m e a Fe(II)
low spin
h e m e a3 Fe(II)
low spin
cyanide added
h e m e a Fe(II)
low spin
h em e a3 Fe(I I )
low spin
sulfide added
h em e a Fe(II)
low spin
h em e a3 Fe(III)
low spin
azide added
1361 c m " 1 1364 c m " 1 1362 c m ' 1 1360 c m 1 1361 c m ' 1 1361 cnf1
1575 c m " 1 1580 c m " 1 1573 c m " 1 1578 c m ' 1
1590 c m ' 1 1590 c m " 1 1590 c m ' 1 1587 c m ' 1 1589 c m ’ 1 1590 cnf1
1620 c m ' 1 1620 c m " 1 1618 c m " 1 1616 c m ’ 1 1616 c m " 1 1620 c m " 1
1630 cm " 1 1630 c m " 1 1630 c m " 1 1628 c m ' 1 1630 c m " 1 1630 c m ' 1
C O
C O
force constants of the porphyrin bonds when the iron is in
the plane of the porphyrin are different from those when
the iron is out of the plane. The change in force con
stants results in shifting of the porphyrin vibrations.
The insensitivity of the spin state vibrations
indicate that heme a3 is not a typical high spin five
coordinate iron porphyrin. The iron of heme a3 is in the
porphyrin plane indicating that the sixth coordination
site of the iron is occupied possibly by a water molecule,
as in the case of myoglobin, or a side group of an amino
acid from the petide backbond of the protein. The side
group would have to be a weak ligand to maintain heme a^
high spin. A good possibility is the carbonyl side group
of an aspartic acid. The carbonyl group could act as a
bridging ligand between the heme a3 and the invisible
copper. The occupation of the sixth coordination of heme
by a carbonyl group in the resting enzyme could explain
the lack of re ac tiv ity of the enzyme to typ ica lly good
heme ligands, carbon monoxide and azide. Upon partial or
fu ll reduction of the enzyme, a conformational change [ 1 2 ]
occurs which moves the carbonyl away and breaks the
coupling between the invisible copper and the heme a^,
giving the resulting high spin iron signal. The sixth
coordination site of heme a is now not occupied and
O
exogenous ligands can be added. After the conformational
change in the enzyme, the heme a3 and Cu^ are s t i l l close
84
enough to constitute a two metal binding site for oxygen.
HISTIDINE
P R O T E IN
HEME
Figure 31. Proposed structure for the active site of the
resting cytochrome oxidase.
Recently spectra of cytochrome oxidase in different
spin and oxidation states were taken by Salmeen et a l . [28].
The published spectra are similar to those spectra reported
here. The authors concluded that the vibrations at 215,
364, 1230 and 1670 cm“^ were due to heme a^. The authors
came to this conclusion on the basis that the vibrations
were present in the reduced enzyme but were absent in the
oxidized enzyme and were not sensitive to the spin state
of heme a^. Contrary to the findings of Salmeen, Table 5
lis ts the vibrations from 200 to 400 cm"'' of reduced
cytochrome oxidase with heme a^ in different spin states.
The vibrations at 216, 275, and 368 cm- ^ do appear to be
sensitive to the spin state of heme a^. I f these vibra
tions are from heme a^, they should not be sensitive to
the spin state of heme a^. The vibrations at 216, 275,
and 358 cm”^ are not from heme a^ but maybe enhanced.
85
vibrational modes from the bridging ligand between a-, and
86
T A B L E 5
List of vibrations in the region from 200 to 400 cm "1.
h em e a Fe(II)
low spin
h em e a3 Fe(I I )
high spin
h em e a Fe(II)
low spin
h e m e a3 Fe(II)
high spin
fluoride added
h em e a Fe(II)
low spin
h em e a3 Fe(II)
high spin
formate added
h em e a Fe(II)
low spin
h em e a, Fe(II)
low spin
cyanide added
h e m e a Fe(II)
low spin
h e m e a3 Fe(II)
low spin
sulfide added
h e m e a Fe(II)
low spin
h e m e a3 Fe(II)
low spin
azide added
216 c m " 1
2 2 0 c m " 1 2 2 0 c m " 1 175 c m " 1 2 2 0 cm "1** 273 cm "1***
275 c m " 1 275 c m " 1 272 c m " 1 242 c m " 1 270 cm "1***
368 c m " 1 370 c m " 1 370 c m " 1 272 cm "1*
* intensity of the vibration m u ch greater as compared to the intensity of the vibration in the high
spin spectrum
** intensity of the vibration reduced greatly as com pared to the intensity of the vibration in the low
spin spectrum
*** intensity of the vibration equal to the intensity of the vibration in the low spin spectrum
00
''V i
SUMMARY
A study of the active site of a metal1oenzyme,
cytochrome oxidase, was made using resonance Raman
spectroscopy. The experimental evidence accumulated
made i t possible to propose a structure for the active
site of cytochrome oxidase.
88
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Asset Metadata

Creator Perreault, Ronald (author)

Core Title Study of cytochrome oxidase by the use of resonance Raman spectroscopy

Contributor Digitized by ProQuest (provenance)

Degree Master of Science

Degree Program Chemistry

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

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-797193

Unique identifier UC11347737

Identifier EP41665.pdf (filename),usctheses-c17-797193 (legacy record id)

Legacy Identifier EP41665.pdf

Dmrecord 797193

Document Type Thesis

Rights Perreault, Ronald

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA