The nitric oxide-bound complexes of reduced yeast cytochrome c oxidase incorporated with [1,3-15N2]histidine have been investigated by EPR spectroscopy. The results of this study have allowed the unambiguous identification of histidine as the endogenous axial ligand to cytochrome alpha 3

Chan, Sunney I.

Stevens, Tom H.

English

Caltech Authors - Main

Communication Vol. 256. No. 3, I s u e  of February 10. pp 1063-1071, 1981 THE J O U R N A L  OF BIOLOGICAL CHEMISTRY Printed m U S.A. 
Histidine Is the  Axial  Ligand to 
Cytochrome u3 in Cytochrome c 
Oxidase* 
(Received  for  publication,  October 30,  1980) 
Tom H. Stevens4 and Sunney I. Chang 
From the Division of Chemistry  and  Chemical 
Engineering, The Chemical  Laboratories,  California 
Institute of Technology, Pasadena, California 91125 
The  nitric  oxide-bound  complexes  of  reduced yeast 
cytochrome c oxidase  incorporated  with [1,3-'SNz]his- 
tidine have been investigated by EPR spectroscopy. 
The results of this study have allowed the  unambiguous 
identification of histidine as the  endogenous  axial lig- 
and to cytochrome u3. 
There  has been  considerable recent  interest  in  the  structure 
of the  cytochrome u3-Cua:, site in cytochrome c oxidase be- 
cause of the  direct involvement of this binuclear center in the 
4-electron reduction of molecular oxygen to  water (1) in the 
final step of cellular respiration. A  body of data is accumulat- 
ing on  the  structure of this  site (1-3). There is now ample 
evidence that  cytochrome u3 is a  high spin  heme iron  in both 
the oxidized and  reduced  forms of the enzyme,  suggesting that 
the  heme is five-coordinate or  that  the  sixth position is occu- 
pied  by a weak  ligand  which is readily  displaced  by exogenous 
ligands. However, magnetic susceptibility data (4) indicate 
that  cytochrome u3 is strongly antiferromagnetically  coupled 
to  CuOJ in the oxidized enzyme to give a S = 2 ground state 
which is EPR-silent. A number of proposals has emerged on 
the nature of the ligand mediating this antiferromagnetic 
coupling between  the  two  metal  centers. One of these propos- 
als (5) involves a bridging imidazole from a histidine residue. 
An endogenous nitrogen atom is indeed implicated in NO- 
binding studies (6, 7) of ferrocytochrome u3 in the reduced 
enzyme.  While the  EPR  data suggest that  this  nitrogen  may 
be  part of a strong  r-donating ligand (8), possibly an imidazole 
from  the  histidine  residue (6), its  identity  has  not been un- 
ambiguously determined. 
The  study described here is to identify the axial  ligand of 
cytochrome u3. One approach  to  this problem is to  prepare 
cytochrome c oxidase with "N-labeled amino acids and  un- 
dertake NO-binding studies  to  the  ferrocytochrome u3 in the 
labeled  enzyme. Inasmuch as an imidazole of histidine  seems 
* This work was supported by Grant GM-22432 from the  National 
Institute of General  Medical Sciences, United States Public Health 
Service, by BRSG  Grant RR07003 awarded by the Biomedical Re- 
search Support Grant Program, Division of Research Resources, 
National Institutes of Health, and by National Research Service 
Award 1T32 GM-07616 from the  National  Institute of General  Med- 
ical Sciences. Contribution No. 6334 from the  Arthur Amos Noyes 
Laboratory of Chemical  Physics, California Institute of Technology, 
Pasadena, Calif. 91125. The costs of publication of this article were 
defrayed  in part by the  payment of page  charges. This article must 
therefore be  hereby  marked  "advertisement"  in  accordance  with 18 
U.S.C. Section 1734 solely to indicate this fact. 
ifornia, Berkeley, Calif. 94720. 
+ Present address, Department of Biochemistry,  University of Cal- 
To whom reprint  requests should be sent. 
to be the most likely candidate for the fifth ligand of the 
cytochrome u3, we have incorporated "N-labeled histidine 
into yeast cytochrome c oxidase. The results of this work 
reveal  that  the axial  ligand is indeed  a  histidine  residue  in the 
yeast enzyme. 
MATERIALS AND METHODS 
The yeast strain Saccharomyces cereuisine D273-10B was muta- 
genized by treatment with ethylmethanesulfonate and a nonpetite 
histidine auxotroph was isolated and purified by standard techniques 
(9). This histidine  auxotroph was grown in a 350-liter fermentor in 
the following media: yeast  nitrogen  base (without  amino acids)  media 
(10); uracil and adenine, 11 g each all L-amino acids  except  histidine, 
17.5 g; ~~-[1,3-''N~]histidine, 95% "N-labeled in both ring positions 
(Vebberlin-Chemie, Germany), 2 g; galactose, 1%; penicillin, 11 g; 
streptomycin, 17 g. The yield of wet yeast cells was about 300 g, of 
which less than 0.004% had reverted to wild type. 
Yeast mitochondria were isolated from the yeast cells by the 
method of Tzagoloff ( l l ) ,  as modified by Shakespeare  and Mahler 
(12). The mitochondrial pellet was resuspended in 10 mM 4-(2-hy- 
droxyethy1)-1-piperazineethanesulfonic acid, 70 m~ sucrose, 110 mM 
mannitol, 1 m~ EDTA,  pH 7.4, and centrifuged at 100,ooO X g. The 
resultant pellet was then resuspended in the  same buffer to a  protein 
concentration of  40-60 mg/ml. This  concentrated  mitochondnal sus- 
pension was used for subsequent  EPR experiments. The wild type 
D273-10B yeast mitochondria were obtained precisely as described 
above  except that no labeled  histidine was added  to  the fermentor. 
Beef heart cytochrome c oxidase was isolated by the procedure of 
Hartzell and  Beinert (13). The purified protein (8.5 nmol of heme a /  
mg of protein) was dissolved in 0.5% Tween 20, 50 mM Tris/HNO.r, 
pH 7.4, to a  protein  concentration of  37 mg/ml. 
The  EPR  samples were prepared by the addition of p-phenylene- 
diamine and  ascorbate to  the anaerobic  samples in EPR  tubes  to final 
concentrations of 2 and 20 m ~ ,  respectively. The NO-bound  species 
were generated by the addition of either NaI4N02  or NaI5NOz (Pro- 
chem, 99% "N) to  these samples (3) to a final concentration of about 
20 mM. It  has been found that  the addition of NOZ- to  p-phenylene- 
diamine- and ascorbate-reduced mitochondrial or the purified re- 
duced (3) protein  results in the observation of EPR signals  from the 
NO-bound ferrocytochrome a 3  of the enzyme almost exclusively. 
Samples of the reduced oxidase, or reduced mitochondrial suspen- 
sions, were allowed to  incubate with  NOZ-  for 30 min at 277 K under 
argon  before being frozen at  77 K. The  EPR  spectra were recorded on 
a  Varian  E-line Century Series  X-Band EPR spectrometer. 
All of the yeast NO-bound spectra were recorded on mitochondrial 
suspensions. Since the concentration of cytochrome oxidase in the 
yeast mitochondrial  samples was considerably lower than  that in the 
purified beef heart cytochrome oxidase samples, it was necessary to 
collect multiple  scans of the mitochondrial  samples to  obtain com- 
parable signal to noise. In addition, background cavity signals plus 
small mitochondrial iron-sulfur protein signals were removed by 
subtracting 10 scans of reduced mitochondria from 10 scans of a 
sample containing  reduced  mitochondria  plus NOz- on a  Spex 
SCAMP SC-32 data processor. 
RESULTS  AND  DISCUSSION 
The binding of NO to  ferrous  hemoproteins is well known 
(14). In  the case of fully reduced  cytochrome c oxidase, NO 
binds  to  cytochrome u3 to  produce  an NO-ferrocytochrome c 
oxidase complex, which exhibits  an  EPR signal rich  in  hyper- 
fine structure. The EPR spectra of 14NO- and I5NO-bound 
beef heart ferrocytochrome c oxidase are shown in Fig. 1. 
When I4NO is bound to ferrocytochrome u3, the  EPR signal 
of the complex exhibits a  nine-line  hyperfine pattern, which 
can  be  interpreted in terms of the  superposition of three sets 
' C. Martin  and S. I. Chan, unpublished  results. 
1069 
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1070 [15N/Histidine-labeled  Yeast Cytochrome c Oxidase 
of three lines arising from two nonequivalent nitrogens ( I  = 
1) interacting with the unpaired electron (6, 7). The larger of 
the two hyperfine coupling constants is 20.3 G and the smaller 
6.8 G.  When 15N0 is used in this experiment, the "NO-bound 
protein exhibits an  EPR spectrum with g values identical with 
those of the 14NO-bound species, but with a hyperfine pattern 
consisting of two sets of three lines (7). This  pattern is con- 
BEEF HEART CYTOCHROME E OXIDASE-'4N0 
A 
203G 
&= 2.09 g, f2.006 g/= 1.97 
BEEF HEART CYTOCHROME E OXIDASE- "NO 
t 
" V  28.20 
t f  
g,=2.09 gz = ' k C 6  gy= 1.97 
FIG. 1. EPR spectra of NO-bound reduced beef heart cyto- 
chrome c oxidase with "NO ( A )  and "NO (B) ,  prepared by the 
addition of either 14NOa- or "NOz- to 0.16 mM cytochrome c 
oxidase. Conditions: temperature, 50 K ,  microwave power, 5 milli- 
watts; modulation amplitude, 2 G microwave frequency, 9.23 GHz. 
sistent with the presence of one 14N and one 15N nitrogen 
bound  axially to cytochrome a3, with a 28.2 G splitting for the 
15N and a 6.8 G splitting for the I4N ligand. The observed 
increase of the larger hyperfine splitting from 20.3 G to 28.2 G 
upon substitution of 15N0  for I4NO is expected  on the basis of 
the relative magnetogyric ratios of the two nitrogen isotopes. 
These observations allow one to assign the larger of the 
hyperfine coupling constants to  the nitrogen of the bound NO 
and the smaller coupling constant to a nitrogen on an endog- 
enous axial  ligand of cytochrome a3. 
In Fig. 2, the  EPR spectra of the normal NO-bound yeast 
protein are compared with those from the purified beef heart 
protein. Aside  from the differences in signal to noise, the  EPR 
parameters for the NO-bound beef heart and yeast proteins 
are essentially identical. 
The  EPR spectra of  I4NO- and "NO-bound  [1,3-"N~]histi- 
dine-substituted yeast cytochrome c oxidase are shown in Fig. 
3. In contrast  to  the [14N]histidine  NO-bound protein spectra, 
the hyperfine patterns in the [1,3-'5Nz]histidine NO-bound 
protein have been altered. The [1,3-"Nz]histidine  "NO-bound 
protein hyperfine pattern consists of two sets of doublets, with 
a 15N0 nitrogen splitting of  27.5 G and a splitting of about 12 
G for the [I5N]nitrogen of histidine. The [1,3-'5N~]histidine 
14NO-bound protein hyperfine pattern consists of three  sets of 
doublets, with splittings of 21 G and 10.2 G for the 14N0 and 
histidine ["N]nitrogens,  respectively. Thus, the substitution 
of [1,3-''Nz]histidine  for  ["Nlhistidine in cytochrome c oxi- 
dase results in the involvement of a ( I  = %) "N nucleus rather 
than a ( I  = 1) 14N nucleus in the NO-bound ferrocytochrome 
a3 EPR signal. The increase of the smaller hyperfine splitting 
from about 7 G to 10 G is expected on the basis of the relative 
magnetogyric ratios of the two nitrogen isotopes. 
In summary, NO binding studies with [1,3-"N~]histidine- 
substituted cytochrome c oxidase have allowed the unequiv- 
ocal identification of histidine as  the endogenous fifth ligand 
to cytochrome a3 in the yeast enzyme. Although this study 
does not address the question of the imidazole  bridge, recent 
work from our laboratory (2, 3) has established that this 
imidazole does not bridge the two metal centers at  the cyto- 
chrome a3-Cu,, site. Thus,  the histidine imidazole is the distal 
axial  ligand of cytochrome a3 vis-U-uis Cu,:,. 
YEAST CYTOCHROME E OXIDASE -"NO "N2-his YEAST Cy~0u.1- ~ I ~ s E . ~ ~  A 
A FIG. 2 ( lef t ) .  EPR spectra of NO- 
bound reduced yeast cytochrome c 
oxidase with 14N0 ( A )  and "NO ( B ) .  
The samples were prepared by the ad- 
dition of either 14N02- or 15N02- to p-  
phenylenediamine- and ascorbate-re- 
duced yeast mitochondria. Conditions: 
temperature, 30 K, microwave  power, 2 
milliwatts; modulation amplitude, 2 G; 
FIG. 3 (right). EPR spectra of NO- 
bound [1,3-"Nz]histidine-substi- 
YEAST CYTOCHROME E. OXDASE-'"NO '%his YEAST CYTOCHROME P OXIDASE-'~NO tuted reduced yeast cytochrome c 
oxidase with I4NO ( A )  and "NO (B) ,  
prepared by the addition of either 
I4NOa- or "NO2- to p-phenylenedia- 
"Nz]histidine-labeled yeast mito- 
chondria. Conditions: temperature, 30 
K microwave  power,  2 milliwatts; mod- 
ulation amplitude, 4 G microwave fre- 
quency, 9.21 GHz. 
4. = 2.09 %=2m6 gy:1.97 microwave frequency, 9.21 GHz. 
t mine- and ascorbate-reduced [lf- 
f 
10.7G - 140 
1 
.J,=ZD9 
t 
g,=zncs pJl.9, gy2.09 
=%e=!- 
92,'2DX 9,:1.97 
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f”NN]Histidine-labeled Yeast Cytochrome c Oxidase 1071 
REFERENCES 
1. Malmstrom, B. G. (1979) Biochim. Biophys. Acta 549, 281-303, 
2. Stevens, T. H., Brudvig, G. W., Bocian, D. F., and Chan, S. I. 
3. Brudvig, G. W., Stevens, T. H., and  Chan, S .  I. (1980) Biochern- 
4. Tweedle, M.  F., Wilson, L. J., Garcia-Ifiiguez, L., Babcock, G.  T., 
5. Palmer, G., Babcock, G. T.,  and Vickery, L. E. (1976) Proc. Natl. 
6. Blokzijl-Homan, M. F. J., and Van Gelder,  B. F. (1971) Biochim. 
and references  cited therein. 
(1979) Proc. Natl.  Acad. Sci. U. S. A .  76,3320-3324 
istry 19,5275-5285 
and Palmer, G.  (1978) J. Biol. Chem. 253,8065-8071 
Acad.  Sci. U. S. A .  73,2206-2210 
Biophys. Acta 234,493-498 
7. Stevens, T. H., Bocian, D. F., and  Chan, S. I. (1979) FEBS Lett. 
8. Kon, H., and  Kataoka, N. (1969) Biochemistry 8,4757-4762 
9. Fink, G. R. (1970) Methods  Enzymol. 17,59-78 
97,314-316 
10. Difco Manual (1953) p. 251, Difco Laboratories, Detroit 
11. Tzagoloff, A. (1969) J. Biol.  Chem. 244,5020-5026 
12. Shakespeare, P. G., and Mahler,  H. R. (1971) J. Biol. Chem. 246, 
13. Hartzell, C. R., and Beinert, H. (1974) Biochirn. Biophys. Acta 
14. Yonetani, T., Yamamoto, H., Erman,  J. E., Leigh, J. R., Jr.,  and 
7649-7655 
368,318-338 
Reed, G. H. (1972) J. Biol. Chem. 247, 2447-2455 
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Histidine is the axial ligand to cytochrome alpha 3 in cytochrome c oxidase

Communication Vol. 256. No. 3, I s u e  of February 10. pp 1063-1071, 1981 THE J O U R N A L  OF BIOLOGICAL CHEMISTRY Printed m U S.A. 
Histidine Is the  Axial  Ligand to 
Cytochrome u3 in Cytochrome c 
Oxidase* 
(Received  for  publication,  October 30,  1980) 
Tom H. Stevens4 and Sunney I. Chang 
From the Division of Chemistry  and  Chemical 
Engineering, The Chemical  Laboratories,  California 
Institute of Technology, Pasadena, California 91125 
The  nitric  oxide-bound  complexes  of  reduced yeast 
cytochrome c oxidase  incorporated  with [1,3-'SNz]his- 
tidine have been investigated by EPR spectroscopy. 
The results of this study have allowed the  unambiguous 
identification of histidine as the  endogenous  axial lig- 
and to cytochrome u3. 
There  has been  considerable recent  interest  in  the  structure 
of the  cytochrome u3-Cua:, site in cytochrome c oxidase be- 
cause of the  direct involvement of this binuclear center in the 
4-electron reduction of molecular oxygen to  water (1) in the 
final step of cellular respiration. A  body of data is accumulat- 
ing on  the  structure of this  site (1-3). There is now ample 
evidence that  cytochrome u3 is a  high spin  heme iron  in both 
the oxidized and  reduced  forms of the enzyme,  suggesting that 
the  heme is five-coordinate or  that  the  sixth position is occu- 
pied  by a weak  ligand  which is readily  displaced  by exogenous 
ligands. However, magnetic susceptibility data (4) indicate 
that  cytochrome u3 is strongly antiferromagnetically  coupled 
to  CuOJ in the oxidized enzyme to give a S = 2 ground state 
which is EPR-silent. A number of proposals has emerged on 
the nature of the ligand mediating this antiferromagnetic 
coupling between  the  two  metal  centers. One of these propos- 
als (5) involves a bridging imidazole from a histidine residue. 
An endogenous nitrogen atom is indeed implicated in NO- 
binding studies (6, 7) of ferrocytochrome u3 in the reduced 
enzyme.  While the  EPR  data suggest that  this  nitrogen  may 
be  part of a strong  r-donating ligand (8), possibly an imidazole 
from  the  histidine  residue (6), its  identity  has  not been un- 
ambiguously determined. 
The  study described here is to identify the axial  ligand of 
cytochrome u3. One approach  to  this problem is to  prepare 
cytochrome c oxidase with "N-labeled amino acids and  un- 
dertake NO-binding studies  to  the  ferrocytochrome u3 in the 
labeled  enzyme. Inasmuch as an imidazole of histidine  seems 
* This work was supported by Grant GM-22432 from the  National 
Institute of General  Medical Sciences, United States Public Health 
Service, by BRSG  Grant RR07003 awarded by the Biomedical Re- 
search Support Grant Program, Division of Research Resources, 
National Institutes of Health, and by National Research Service 
Award 1T32 GM-07616 from the  National  Institute of General  Med- 
ical Sciences. Contribution No. 6334 from the  Arthur Amos Noyes 
Laboratory of Chemical  Physics, California Institute of Technology, 
Pasadena, Calif. 91125. The costs of publication of this article were 
defrayed  in part by the  payment of page  charges. This article must 
therefore be  hereby  marked  "advertisement"  in  accordance  with 18 
U.S.C. Section 1734 solely to indicate this fact. 
ifornia, Berkeley, Calif. 94720. 
+ Present address, Department of Biochemistry,  University of Cal- 
To whom reprint  requests should be sent. 
to be the most likely candidate for the fifth ligand of the 
cytochrome u3, we have incorporated "N-labeled histidine 
into yeast cytochrome c oxidase. The results of this work 
reveal  that  the axial  ligand is indeed  a  histidine  residue  in the 
yeast enzyme. 
MATERIALS AND METHODS 
The yeast strain Saccharomyces cereuisine D273-10B was muta- 
genized by treatment with ethylmethanesulfonate and a nonpetite 
histidine auxotroph was isolated and purified by standard techniques 
(9). This histidine  auxotroph was grown in a 350-liter fermentor in 
the following media: yeast  nitrogen  base (without  amino acids)  media 
(10); uracil and adenine, 11 g each all L-amino acids  except  histidine, 
17.5 g; ~~-[1,3-''N~]histidine, 95% "N-labeled in both ring positions 
(Vebberlin-Chemie, Germany), 2 g; galactose, 1%; penicillin, 11 g; 
streptomycin, 17 g. The yield of wet yeast cells was about 300 g, of 
which less than 0.004% had reverted to wild type. 
Yeast mitochondria were isolated from the yeast cells by the 
method of Tzagoloff ( l l ) ,  as modified by Shakespeare  and Mahler 
(12). The mitochondrial pellet was resuspended in 10 mM 4-(2-hy- 
droxyethy1)-1-piperazineethanesulfonic acid, 70 m~ sucrose, 110 mM 
mannitol, 1 m~ EDTA,  pH 7.4, and centrifuged at 100,ooO X g. The 
resultant pellet was then resuspended in the  same buffer to a  protein 
concentration of  40-60 mg/ml. This  concentrated  mitochondnal sus- 
pension was used for subsequent  EPR experiments. The wild type 
D273-10B yeast mitochondria were obtained precisely as described 
above  except that no labeled  histidine was added  to  the fermentor. 
Beef heart cytochrome c oxidase was isolated by the procedure of 
Hartzell and  Beinert (13). The purified protein (8.5 nmol of heme a /  
mg of protein) was dissolved in 0.5% Tween 20, 50 mM Tris/HNO.r, 
pH 7.4, to a  protein  concentration of  37 mg/ml. 
The  EPR  samples were prepared by the addition of p-phenylene- 
diamine and  ascorbate to  the anaerobic  samples in EPR  tubes  to final 
concentrations of 2 and 20 m ~ ,  respectively. The NO-bound  species 
were generated by the addition of either NaI4N02  or NaI5NOz (Pro- 
chem, 99% "N) to  these samples (3) to a final concentration of about 
20 mM. It  has been found that  the addition of NOZ- to  p-phenylene- 
diamine- and ascorbate-reduced mitochondrial or the purified re- 
duced (3) protein  results in the observation of EPR signals  from the 
NO-bound ferrocytochrome a 3  of the enzyme almost exclusively. 
Samples of the reduced oxidase, or reduced mitochondrial suspen- 
sions, were allowed to  incubate with  NOZ-  for 30 min at 277 K under 
argon  before being frozen at  77 K. The  EPR  spectra were recorded on 
a  Varian  E-line Century Series  X-Band EPR spectrometer. 
All of the yeast NO-bound spectra were recorded on mitochondrial 
suspensions. Since the concentration of cytochrome oxidase in the 
yeast mitochondrial  samples was considerably lower than  that in the 
purified beef heart cytochrome oxidase samples, it was necessary to 
collect multiple  scans of the mitochondrial  samples to  obtain com- 
parable signal to noise. In addition, background cavity signals plus 
small mitochondrial iron-sulfur protein signals were removed by 
subtracting 10 scans of reduced mitochondria from 10 scans of a 
sample containing  reduced  mitochondria  plus NOz- on a  Spex 
SCAMP SC-32 data processor. 
RESULTS  AND  DISCUSSION 
The binding of NO to  ferrous  hemoproteins is well known 
(14). In  the case of fully reduced  cytochrome c oxidase, NO 
binds  to  cytochrome u3 to  produce  an NO-ferrocytochrome c 
oxidase complex, which exhibits  an  EPR signal rich  in  hyper- 
fine structure. The EPR spectra of 14NO- and I5NO-bound 
beef heart ferrocytochrome c oxidase are shown in Fig. 1. 
When I4NO is bound to ferrocytochrome u3, the  EPR signal 
of the complex exhibits a  nine-line  hyperfine pattern, which 
can  be  interpreted in terms of the  superposition of three sets 
' C. Martin  and S. I. Chan, unpublished  results. 
1069 
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1070 [15N/Histidine-labeled  Yeast Cytochrome c Oxidase 
of three lines arising from two nonequivalent nitrogens ( I  = 
1) interacting with the unpaired electron (6, 7). The larger of 
the two hyperfine coupling constants is 20.3 G and the smaller 
6.8 G.  When 15N0 is used in this experiment, the "NO-bound 
protein exhibits an  EPR spectrum with g values identical with 
those of the 14NO-bound species, but with a hyperfine pattern 
consisting of two sets of three lines (7). This  pattern is con- 
BEEF HEART CYTOCHROME E OXIDASE-'4N0 
A 
203G 
&= 2.09 g, f2.006 g/= 1.97 
BEEF HEART CYTOCHROME E OXIDASE- "NO 
t 
" V  28.20 
t f  
g,=2.09 gz = ' k C 6  gy= 1.97 
FIG. 1. EPR spectra of NO-bound reduced beef heart cyto- 
chrome c oxidase with "NO ( A )  and "NO (B) ,  prepared by the 
addition of either 14NOa- or "NOz- to 0.16 mM cytochrome c 
oxidase. Conditions: temperature, 50 K ,  microwave power, 5 milli- 
watts; modulation amplitude, 2 G microwave frequency, 9.23 GHz. 
sistent with the presence of one 14N and one 15N nitrogen 
bound  axially to cytochrome a3, with a 28.2 G splitting for the 
15N and a 6.8 G splitting for the I4N ligand. The observed 
increase of the larger hyperfine splitting from 20.3 G to 28.2 G 
upon substitution of 15N0  for I4NO is expected  on the basis of 
the relative magnetogyric ratios of the two nitrogen isotopes. 
These observations allow one to assign the larger of the 
hyperfine coupling constants to  the nitrogen of the bound NO 
and the smaller coupling constant to a nitrogen on an endog- 
enous axial  ligand of cytochrome a3. 
In Fig. 2, the  EPR spectra of the normal NO-bound yeast 
protein are compared with those from the purified beef heart 
protein. Aside  from the differences in signal to noise, the  EPR 
parameters for the NO-bound beef heart and yeast proteins 
are essentially identical. 
The  EPR spectra of  I4NO- and "NO-bound  [1,3-"N~]histi- 
dine-substituted yeast cytochrome c oxidase are shown in Fig. 
3. In contrast  to  the [14N]histidine  NO-bound protein spectra, 
the hyperfine patterns in the [1,3-'5Nz]histidine NO-bound 
protein have been altered. The [1,3-"Nz]histidine  "NO-bound 
protein hyperfine pattern consists of two sets of doublets, with 
a 15N0 nitrogen splitting of  27.5 G and a splitting of about 12 
G for the [I5N]nitrogen of histidine. The [1,3-'5N~]histidine 
14NO-bound protein hyperfine pattern consists of three  sets of 
doublets, with splittings of 21 G and 10.2 G for the 14N0 and 
histidine ["N]nitrogens,  respectively. Thus, the substitution 
of [1,3-''Nz]histidine  for  ["Nlhistidine in cytochrome c oxi- 
dase results in the involvement of a ( I  = %) "N nucleus rather 
than a ( I  = 1) 14N nucleus in the NO-bound ferrocytochrome 
a3 EPR signal. The increase of the smaller hyperfine splitting 
from about 7 G to 10 G is expected on the basis of the relative 
magnetogyric ratios of the two nitrogen isotopes. 
In summary, NO binding studies with [1,3-"N~]histidine- 
substituted cytochrome c oxidase have allowed the unequiv- 
ocal identification of histidine as  the endogenous fifth ligand 
to cytochrome a3 in the yeast enzyme. Although this study 
does not address the question of the imidazole  bridge, recent 
work from our laboratory (2, 3) has established that this 
imidazole does not bridge the two metal centers at  the cyto- 
chrome a3-Cu,, site. Thus,  the histidine imidazole is the distal 
axial  ligand of cytochrome a3 vis-U-uis Cu,:,. 
YEAST CYTOCHROME E OXIDASE -"NO "N2-his YEAST Cy~0u.1- ~ I ~ s E . ~ ~  A 
A FIG. 2 ( lef t ) .  EPR spectra of NO- 
bound reduced yeast cytochrome c 
oxidase with 14N0 ( A )  and "NO ( B ) .  
The samples were prepared by the ad- 
dition of either 14N02- or 15N02- to p-  
phenylenediamine- and ascorbate-re- 
duced yeast mitochondria. Conditions: 
temperature, 30 K, microwave  power, 2 
milliwatts; modulation amplitude, 2 G; 
FIG. 3 (right). EPR spectra of NO- 
bound [1,3-"Nz]histidine-substi- 
YEAST CYTOCHROME E. OXDASE-'"NO '%his YEAST CYTOCHROME P OXIDASE-'~NO tuted reduced yeast cytochrome c 
oxidase with I4NO ( A )  and "NO (B) ,  
prepared by the addition of either 
I4NOa- or "NO2- to p-phenylenedia- 
"Nz]histidine-labeled yeast mito- 
chondria. Conditions: temperature, 30 
K microwave  power,  2 milliwatts; mod- 
ulation amplitude, 4 G microwave fre- 
quency, 9.21 GHz. 
4. = 2.09 %=2m6 gy:1.97 microwave frequency, 9.21 GHz. 
t mine- and ascorbate-reduced [lf- 
f 
10.7G - 140 
1 
.J,=ZD9 
t 
g,=zncs pJl.9, gy2.09 
=%e=!- 
92,'2DX 9,:1.97 
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f”NN]Histidine-labeled Yeast Cytochrome c Oxidase 1071 
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Biochim. and references cited therein.

Methods Enzymol.

Methods Enzymol. 17,59-78 97,314-316 10. Difco Manual

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https://authors.library.caltech.edu/11604/1/STEjbc81.pdf

Histidine is the axial ligand to cytochrome alpha 3 in cytochrome c oxidase

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