Invited Cytochrome MARIA Department

of Biochemistry

c Oxidase:


and Biophysics, Received








of Pennsylvania,

15, 1977; revised





18, 1977

under different conditions of SDS”-gel electrophoresis and this, in part, may explain the different subunit structures that have been reported for this complex. In S. cereuisiae, all seven subunits exhibit acidic isoelectric points (4); subunits syncytoplasmic ribosomes thesized on (IV-VII) are more acidic than those (I-III) coded by the mitochondrial genome. Amino acid analysis of isolated subunits showed that polypeptides I and III were hydrophobic while polypeptides II and IV-VI contained about 50% polar amino acids. Similar results (Table I) were obtained for the isolated subunits of the beef heart and N. crassa cytochrome c oxidase. To explore the arrangement of these subunits in the holoenzyme, the reactivity of each subunit with a variety of “surface probes” was tested with isolated cytochrome c oxidase, with cytochrome c oxidase incorporated into liposomes, and with mitochondrially bound oxidase. The surface probes included iodination with lactoperoxidase and coupling with the memreagent p-diazonium brane-impermeant benzene-sulfonate (13, 14). In addition, external subunits were identified by linking them to bovine serum albumin carrying a covalently bound tolylene-2,4-diisocyanate group (15). With mitochondria, subunits II, V, and VI were labeled by iodination and by reacting with radioactive p-diazonium benzene-[35S]sulfonate, whereas in purified submitochondrial particles most of the label was in subunit III. With purified oxidase, subunits I and IV were inaccessible to those reagents, whereas the other four were accessible. It was concluded that the ar-

This paper is intended to provide a short, updated summary of experimental work on cytochrome c oxidase which has been carried out since early 1974. For a discussion of earlier investigations, the reader should consult the original papers and historical reviews (for example, Refs. l-3). The emphasis and interpretations presented here reflect the authors’ own experience and judgment. Throughout the paper, we have tried, however, to indicate the views of other investigators especially when they were contrasted with our own. References are given to publications where these views are expressed so that the reader may consult them and form his own opinion, For consistency of presentation, the nomenclature for cytochromes a and as follows the “classic” definition in which two cytochromes represent two different components of the oxidase. The controversy regarding this concept is discussed in the final section of the review. SUBUNIT

A Synopsis’


It has been generally agreed that cytochrome oxidase isolated from various sources such as beef heart, yeast (Saccharomyces cereuisiae), and fungus (Neurospora crassa) is composed of seven subunits (4-8). In the case of the beef heart preparation, the subunit composition of the cytochrome c oxidase complex (6) is independent of the isolation procedure (g-11). It was pointed out recently (12) that the order of migration of polypeptides varies ’ Supported by Grants GM 21524, GM 12202, and 18708 from the National Institutes of Health. ’ M. E. is an American Heart Established Investigator. HL

” Abbreviations used: SDS, sodium dodecyl sulfate; DEAE, dietbylaminoethyl; IgG, immunoglobulin G; epr, electron paramagnetic resonance.


0003-9861/78/1881-0015$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.




Beef heart

S. cerevisiae N. crassa






35.5 24.7 32.5

44.7 42.1 40.1

39.9 35.8

6 4 5

o The nomenclature of the subunits referred to in this table and throughout this review is that used by the authors of the original papers.

rangement of cytochrome c oxidase in the inner mitochondrial membrane is transmembranous and asymmetric; subunits II, V, and VI are situated on the outer side, subunit III is situated on the matrix side, and subunits I and IV are buried in the interior of the membrane. Moreover, in the presence of cytochrome c, conjugation of bovine serum albumin carrying an isocyanate group with subunits V and VI was inhibited, which led to the suggestion that they were involved in the binding of cytochrome c (15). A similar conclusion was reached on the basis of recent studies with photoaffinity-labeled cytochrome c bound to the specific cytochrome c binding site on the inner mitochondrial membrane. After irradiation of these mitochondria, fractionation in the presence of salts and detergents resulted in the recovery of a cytochrome c-cytochrome oxidase complex (16). Electrophoresis on polyacrylamide gels in the presence of SDS showed that the two smallest subunits of the oxidase were the site of interaction with cytochrome c. Another attempt to determine the near-neighbor relationships of the subunits of cytochrome c oxidase involved the use of cross-linking reagents (17). Many products were formed and clear-cut conclusions were difficult to draw. Subunit-specific antisera (18) were prepared to assess the role of individual subunits of the yeast cytochrome c oxidase in enzymatic activity and to determine by immunological means the extent to which the seven polypeptides of the holoenzyme were physically associated. An antiserum against one mitochondrially made subunit and antisera against all four cytoplasmically made subunits were capable of inhibiting the oxidation of ferrocytochrome c by cytochrome



c oxidase. An antiserum which cross-reacted with only a single subunit precipitated all seven cytochrome c oxidase subunits from crude mitochondrial extracts. It was suggested (18) that both mitochondrially made and cytoplasmically made subunits contributed to the enzymatic function of cytochrome c oxidase and that all seven polypeptides were tightly associated. In contrast to this, several observations were reported (19-21) which suggested that cytochrome c oxidase containing only the smaller subunits retains full enzymatic activity in oxidizing ferrocytochrome c. Phan and Mahler (19) prepared such “modified” cytochrome c oxidase from yeast S. cereuisiae and beef heart mitochondria by means of apolar chromatography on L-leucine coupled to agarose. It lacked the two largest subunits ( I and II) and contained minimal amounts of subunit III. The heme and copper content of this enzyme was >20 nmol/mg of protein and the minimal molecular weight was 50,000. Komai and Capaldi (20) purified cytochrome c oxidase in a medium containing potassium cholate and ammonium sulfate to a heme a content of 14.6 nmol/mg of protein and reported that the enzyme had only two molecular weight species of polypeptide of 14,000 and 11,500 and was catalytically active. The activity was, however, extremely low, which suggests that these subunits may have been decomposition products rather than constituents of an active enzyme. A similar preparation was described by Yamamoto and Orii (21), who used controlled digestion with chymotrypsin (“proteinase-treated cytochrome oxidase”). Recently, the purification of a subunit which contains 40 nmol of heme a/mg of protein has been reported (22). It involves a reaction of cytochrome c oxidase with pyridine, precipitation by acidification, and chromatography on a DEAE-cellulose column. The purified subunit had a molecular weight of 11,600 and was homogeneous on polyacrylamide gels in the presence of SDS and P-mercaptoethanol. Although all of these results seem to indicate that a minimal number of subunits may indeed be required for electron transfer, one should bear in mind that cytochrome oxidase in mitochondria in situ is also involved in energy coupling and that



perhaps all seven subunits arranged in a very specific way are required for coupled electron flow. It has been recently reported (23) that phospholipid vesicles reconstituted with cytochrome oxidase and capable of high oxidase activity and energy coupling exhibit particles on fracture faces of freeze-fractured membranes whereas control preparations made with phospholipids alone show no particles. Examination of crossfractured vesicle membranes shows the particles to be present in a transmembranous position. As a direct approach to the question of the distribution and orientation of cytochrome c oxidase in the inner mitochondrial membrane, Hackenbrock and Hammon (24) have prepared a highly purified immunoglobulin of the IgG class monospecific for the oxidase. The IgG inhibited succinate oxidase activity and ascorbatecytochrome c oxidase activity immediately and completely when reacted with either a purified, metabolically and structurally intact inner membrane-matrix fraction from rat liver mitochondria or an inverted inner membrane vesicle preparation. Furthermore, the immunoglobulin completely displaced cytochrome c from the surface of the inner membrane-matrix fraction as shown by analysis of ferricyanide reduction and difference spectra. The authors suggested that all cytochrome c oxidase which is active during the oxidation of succinate and ascorbate is accessible on both surfaces of the inner membrane through a transmembrane orientation. It was also shown by using ferritin-conjugated antibody that there were approximately 2000 cytochrome c oxidase binding sites on the outer surface of intact inner membrane in relatively disordered spatial distributions over both cristal and inner boundary membrane regions. This number is, however, smaller than that calculated from the spectral data by various assumptions. ORIENTATION



Initial attempts to detect orientation of cytochrome oxidase within the mitochondrial membrane were made using the method of linear dichroism and photoselection (25). The dichroic ratios on the order of 1.3 were found to be very close to those



expected for a planar chromophore which is “immobilized” with respect to the laboratory axis. Since there was no other relaxation of the photoinduced linear dichroism observed than that attributable to the rotation of mitochondria and submitochondrial particles in suspension, the authors suggested that either cytochrome oxidase was completely immobilized within the mitochondrial membrane or it rotated around one single axis which was parallel to the symmetry axis of the cytochrome a3 heme. To obtain information on the distribution and structure of the cytochrome oxidase molecule in the membrane profile and in the membrane plane an electron diffraction technique (26) and X-ray diffraction method (27) were applied to ordered bilayers of purified membranous cytochrome oxidase. Analyses of electron micrographs taken at different tilt angles showed that cytochrome oxidase molecules were sticking far out into solution on the inside surface of the vesicle. Pairs of molecules were found to be related by a crystallographic twofold axis perpendicular to the plane of the membrane. Analyses of X-ray diffraction patterns obtained for hydrated oriented multilayers of “membraneous” cytochrome c oxidase (IO), formed by centrifugation followed by slow partial dehydration at 4°C (27, 28), led to the following suggestions: (i) The cytochrome oxidase molecules are oriented asymmetrically in the membrane profile. (ii) A significant portion of the oxidase molecule is present within the extravesicular surface of the membrane. (iii) The oxidase molecule extends over the entire thickness of the membrane (i.e., spans the membrane). Moreover, the low angle equatorial diffractions which arise from the packing of oxidase molecules in the plane of the membrane were characteristic of a noncrystalline planar arrangement. However, the high degree of asymmetry in the single membrane profile, and the presence of strong equatorial and meridional diffractions [for details see the original paper (28)], indicated that the cytochrome oxidase molecules were all oriented similarly in the membrane profile with the bundles of cY-helices normal to the plane of the membrane. To determine the orientation of



the heme chromophores with respect to the plane of the membrane, polarized optical absorption and epr spectroscopy were applied to the same oriented oxidase multilayers (Fig. 1). To distinguish between the two hemes, a and as, use was made of the various ligands of the oxidase (azide, sulfide, formate, carbon monoxide) which form spectrally identifiable compounds (29). Both techniques gave consistent results which showed (28, 29) that the two hemes were oriented such that the angle between the normal to the heme plane and the normal to the membrane plane was close to 90”. Moreover, the average orientations of the hemes were qualitatively similar in the oxidized and reduced membranes. Further studies on the oriented hydrated multilayers of mitochondrial membranes (30, 31) demonstrated that in mitochondria in situ the hemes of the oxidase are also oriented with the normal to their heme planes lying approximately in the plane of the mitochondrial membrane. MAGNETIC PROPERTIES OF THE COMPONENTS OF CYTOCHROME c OXIDASE

Measurements of the properties of the hemes and coppers of cytochrome oxidase (32-42) have established that the amount

FIG. 1. Visible absorption spectra of the reduced cytochrome c oxidase in the oriented multilayers of the “membranous” enzyme. The hydrated oriented multilayer was formed as described in Ref. 29 and reduced by the addition of a 1 M ascorbate solution in pH 6.8.



of the low spin ferric heme represented by the signal measured in the fully oxidized hemeprotein is quantitatively equal to the concentration of the one-half of total heme a and corresponds to that of cytochrome a (or ad. This low spin ferric heme signal disappears during reductive titrations under anaerobic conditions while a high spin ferric heme signal appears, attains a maximum, and then disappears (32-37). The disappearance of the low spin heme signal and the appearance of the high spin signal have the same dependence on redox potential and the half-reduction potential values at pH 7.0 and 8.5 are 0.38 and 0.30 V, respectively (35). The high spin heme signal attains an intensity equal to the cytochrome a concentration in intact mitochondria and submitochondrial particles (34,35) but not in preparations of isolated cytochrome oxidase (32, 35). Three of the four components of cytochrome oxidase can be observed by epr when the reduced cytochrome as-NO compound is formed and then cytochrome a and the “visible” copper are reoxidized with limiting amounts of ferricyanide (35). (The copper atom which exhibits an optical absorption maximum at 830 nm and an epr resonance at g = 2 is referred to as the “visible” copper. Its Em7.2value is 0.245 f 0.01 V. The other copper atom remains undetectable in either the optical or epr measurements and thus is referred to as the “invisible” copper.) Under these conditions cytochrome a3 is seen as the ferrocytochrome-NO compound (g = 2), cytochrome a as a low spin ferric heme, and the “visible” copper can be identified by its characteristic g = 2.03 signal. Moreover, the epr spectrum of the cytochrome as-NO compound can be measured with cytochrome a and the “visible” copper either oxidized or reduced. Although interactions were observed by changes in the shape of the signal and in its hyperhne characteristics, there was no significant change in the total amount of the signal as determined by double integration (35). This suggests that neither cytochrome a nor the “visible” copper is close enough (and/or are oriented properly) to cause significant paramagnetic quenching of the epr signal of the reduced cytochrome as-NO compound.



Several lines of evidence indicate that the epr-undetected heme in fully oxidized cytochrome oxidase is high spin. Additon of cyanide causes an absorption change expected for a high spin heme changing to a low spin cyanide compound (38, 39). Magnetic circular dichroism measurements (40-42) also show that one of the two hemes in the fully oxidized cytochrome oxidase is high spin ferric (and one is high spin ferrous in fully reduced cytochrome oxidase). Magnetic susceptibility measurements on samples maintained at room temperature (43) suggested that the high spin ferric heme may be anti-ferromagnetically coupled to an oxidized copper atom because the measured susceptibility was lower than that expected for the total paramagnetic centers present in the preparation. The picture of the magnetic properties of the hemes of cytochrome oxidase which emerges from these results and is most generally agreed upon may be summarized as follows: In fully oxidized cytochrome oxidase, cytochrome a3 is high spin ferric and cytochrome a is low spin ferric; in anaerobic suspensions of half-reduced cytochrome oxidase, cytochrome a3 is low spin ferrous and cytochrome a is high spin ferric; in fully reduced cytochrome oxidase, cytochrome a3 is high spin ferrous and cytochrome a is low spin ferrous. The X-ray absorption edge spectra (EXAFS) of oxidized and reduced preparations of isolated cytochrome c oxidase have been reported recently (44). A comparison of the spectra with those of model compounds led the authors to suggest that in the oxidized heme protein one of the two copper atoms is reduced (Cu’) and the other is oxidized (Cu”). This interesting interpretation should be viewed with caution because the measurements were made at very high protein concentrations and endogenous electron donors present in the cytochrome c oxidase preparation may have caused partial reduction of the samples used in the experiments. Although the authors claim that this is unlikely, adequate experimental evidence was not presented. Reaction of cytochrome c oxidase with CO and other ligands. The reactions of cytochrome c oxidase with different ligands have been the subject of continuing inves-



tigations. Carbon monoxide, because of its competitive nature with respect to oxygen, has been of particular interest. Anaerobic potentiometric titrations of cytochrome oxidase carried out in the presence of high concentrations (up to 1 mu) of CO by Lindsay and Wilson gave an n value of 2.0 for the formation of the CO compound of reduced cytochrome a3 (45, 46). Analysis of the CO concentration dependence of the measured E, value for the formation of the CO compound showed that it became 30 mV more positive with each lo-fold increase in CO concentration (46). It was therefore concluded that carbon monoxide binds with high affinity only when two redox components are reduced: cytochrome a3 (E,,,T.o = 0.385 V) and the “invisible copper.” [It was estimated from potentiometric (45,46) and coulometric (47) titrations that the half-reduction potential for the invisible about 0.340 V.] The copper is oxidation-reduction reactions of cytochrome a3 and the “invisible” copper including control of the reaction of cytochrome oxidase with CO were interpreted according to the schematic diagram shown in Fig. 2. An independent confirmation for the results obtained during potentiometric titrations came from anaerobic reductive titrations with NADH and oxidative titrations with molecular oxygen of both isolated cytochrome oxidase and submitochondrial particles in the presence of CO (48, 49). Results reported by Wever and co-workers (49) and from our own laboratory (48) agree in that 2 eq are required for formation of the reduced cytochrome Q-CO compound. In contrast, Anderson et al. (47) reported that, in the coulometric titrations in the presence of CO, 3 out of 4 eq in isolated cytochrome oxidase could be removed from the preparation without oxidizing the CO compound. It has been observed in many laboratories that when anerobic suspensions of cytochrome oxidase are treated with CO, endogenous electron donors (or oxidation of CO itself) present in the preparation lead to formation of the reduced cytochrome as-CO compound (48-51). This could explain both the failure of Anderson et al. (47) to oxidize the CO compound and the removal of 3 eq per cytochrome a8 from




of the oxidase, which indicates that its formation depends on the reduction of other component(s) of the oxidase. Potentiometric titrations in the presence of azide revealed that the appearance of the g = 2.9 signal required reduction of both the high potential heme and the “invisible copper” (35). Thus, the currently available data suggest that the “invisible copper” plays a key role in the organization of the “active site” of cytochrome oxidase, both by controlling its accessibility to reactants and as a direct participant in the catalytic function. Other ligands which have been the subject of recent reports, particularly those of Nicholls and co-workers (52, 53, 55, 56), include sulfide (35,53,54) and formate (55, 56). Sulfide was shown to form a well-defined inhibitory compound with a Ki of less Kd 06pM than 0.1 PM which exhibits low spin ferric heme epr signals typical of a sulfur compound (g values near 2.6, 2.2, and 1.9). In mitochondrial membranes and some prepN N-{&=0 ;:+arations of isolated cytochrome oxidase each signal is double (g = 2.57 and g = 2.54, 2.26, and 2.22), which suggest the existence of two different species (35). Formate inFIG. 2. A schematic representation of the oxidahibits respiration by forming a high spin tion-reduction reactions of cytochrome 03 and the ferric heme compound of cytochrome a3 “invisible copper”, including control of the r&action of cytochrome oxidase with CO. Only the cytochrome a3 with a Ki of from 1 to 30 mM at pH 7.4, and the “invisible copper” are represented for simplicdepending on experimental conditions (56). ity. The heme is represented as if it were cut through the iron atom by a plane perpendicular to the heme plane and the copper is represented as if it were bound to three or four ligands. The mechanism depicts a major structural rearrangement which occurs when both components are reduced, permitting the reaction with CO. [From Wilson et al. (34); reprinted by permission.]

the preparation. Further experiments will be mandatory to resolve the discrepancy concerning the stoichiometry of reducing equivalents. The “invisible copper” has also been considered as an important participant in the reaction of cytochrome c oxidase with other ligands. The addition of azide causes a small change in the g = 3 epr signal in the fully oxidized system, shifting part of the signal to a lower magnetic field (35), but the g = 2.9 signal typical of the low spin ferric heme azide complex is not observed. The g = 2.9 signal of the low spin heme ferric azide complex does appear on partial reduction





According to Mitchell’s chemiosmotic hypothesis of energy coupling (57), cytochrome oxidase catalyzes transmembrane electron transfer and leads to separation of charges across the mitochondrial membrane. Direct measurements of the electric current generation by cytochrome oxidase carried out either in planar, oxidase-containing membranes or in proteoliposomes showed generation of a transmembrane electric potential difference, positive on the cytochrome c side of the membrane (58). In suspension of these proteoliposomes, electron transfer via cytochrome c was found to be accompanied by the uptake of penetrating ions; tetraphenyl phosphonium cation was coupled with electron transfer via external cytochrome c while electron transfer through intraproteoliposomal cytochrome c induced the uptake of tetraphenyl borate anion. Both the generation of the trans-



membrane electric potential difference and the uptake of penetrating ions were sensitive to addition of cyanide and uncouplers. In a recent short communication (59), Wikstrom reported measurements of H+ translocation across the inner mitochondrial membrane coupled to the redox activity of cytochrome c oxidase. The calculated stoichiometry was 4H+/2e- when ferrocyanide was used as substrate. This result was used as the basis for the postulate that the third phosphorylation site functions as a proton pump with a stoichiometry of 4H+ transported for each two electrons transferred from cytochrome c to oxygen. This would establish a stoichiometry of 4H+/ATP for the reversible ATP-coupled proton pump of the chemiosmotic hypothesis. An examination of the data presented by Wikstrom (59) suggests, however, that considerable additional experiments are required to establish that cytochrome oxidase acts as a proton pump and to determine its stoichiometry. KINETICS OF THE REACTION BETWEEN CYTOCHROME c AND CYTOCHROME c OXIDASE

The kinetics of the reaction between ferrocytochrome c and cytochrome c oxidase in a fully oxidized or mixed-valence state enzyme under both aerobic and anaerobic conditions was studied by the rapid spectrophotometric techniques of stopped flow (60-63) and temperature jump (63). It was found that the very initial reaction was the reduction of cytochrome a by ferrocytochrome c, which occurred with a secondorder rate constant of 2-10 x lo6 M-’ s-l. This initial rapid phase was followed by a slower reaction identified with a transfer of electrons between cytochrome a and the visible copper. The equilibrium constant for the reaction between cytochromes c and a was found to be very close to unity. Kinetic studies on isolated cytochrome oxidase carried out over a range of substrate concentrations (54) suggested that in addition to the usual analysis based on the Michaelis-Menten postulate of productive complex formation, an alternative mechanism involving dead-end complex formation can equally well predict the rate equation as deduced from exneriment.



Using both a kinetic approach (65) and direct binding measurements (66) the presence of two binding sites for cytochrome c per cytochrome c oxidase has been demonstrated for cytochrome c-depleted mitochondrial membranes. Horse heart cytochrome c reacted with these two sites with different affinities (K,i = 4 X lop8 M and KmZ = 1 x lop6 M) while yeast cytochrome c reacted with both sites with equally high affinity (K,,, = 4 X 10e8 M) and euglena cytochrome reacted with equally low affinity (4 X IOe7 M). REACTION OF CYTOCHROME c OXIDASE WITH MOLECULAR OXYGEN

Progress has been made in understanding the reaction of molecular oxygen with cytochrome oxidase. The CO binding data suggest that both cytochrome a3 and the “invisible copper” are involved in the active site of the oxidase. This allowed Wilson and co-workers (67) to put forward a mechawhich can account for the nism oxidation-reduction properties of cytochrome oxidase as well as the kinetics and thermodynamics of the oxygen reaction. Based on the analogy with CO binding (45, 46), this mechanism proposes that oxygen forms a bridged compound between the iron of cytochrome a3 and the “invisible copper” and then is reduced in a two-electron step to form a relatively stable bound peroxide. The bound peroxide is then further reduced to water in either two oneelectron steps or one two-electron step. Since the two-electron reduction of oxygen to bound peroxide has an Ern7.0of near 0.7 V (68, 69) and the reaction in cytochrome oxidase occurs at an Eh of 0.5 to 0.6 V (70) the mechanism overcomes the difficulty imposed by thermodynamic barriers of a oneelectron reduction of oxygen to superoxide anion, which at pH 7.0 has a half-reduction potential of -0.32 V (68, 69). Further kinetic attempts were made to study the mechanism of cytochrome oxidase and possible intermediates in the reaction. Following mixing of oxygen and suspensions of reduced isolated cytochrome oxidase at room temperature, Orii and King (71) observed rapid formation of an intermediate with an absorption maximum near 428 nm (“oxygenated” cytochrome oxidase)



which was converted to the fully oxidized form in a reaction catalyzed by cytochrome c. The latter occurred in three phases indicating heterogeneity in the reaction products, but the transitions were rapid enough at high cytochrome c concentration to be possible intermediates in the catalytic reaction cycle. Greenwood and co-workers (72) measured the rate of reaction of molecular oxstate cytoygen with “mixed-valence” chrome oxidase prepared by partial oxidation with ferricyanide of the CO compound of reduced cytochrome oxidase. The reaction rate was followed after the addition of 02 and photolysis of the CO compound. In agreement with the earlier work of Chance and co-workers (73)) oxygen reacted as rapidly with the partially reduced oxidase (8 x lo7 M-’ s-l) as with the fully reduced enzyme. The reaction product for the reaction of oxygen with the partially reduced cytochrome oxidase was “oxygenated” oxidase characterized by a 611-nm absorption maximum and not the fully oxidized enzyme. Chance and co-workers (74-76) introduced kinetic measurements at low temperatures on preparations suspended in ethylene glycol-water mixtures for studying the cytochrome oxidase-oxygen reaction. This method involves preparing the reduced cytochrome oxidase-CO compound in the liquid phase at temperatures low enough so that CO does not exchange for oxygen at a significant rate. The sample is then mixed with oxygen, frozen, and brought to the desired temperature. The reaction with oxygen is initiated by flash photolysis of the CO compound. The first reaction product formed (termed compound A) is accompanied by the appearance of a maximum at SO-591 nm and a trough at 611 nm (difference spectrum with respect to the reduced cytochrome oxidase). This spectrum was interpreted as arising from the formation of an oxygen compound of reduced cytochrome a3 on the basis of its similarity to the CO compound (Fig. 3). Its postulated formal Valency was Cu+a32+-02. The second-order rate constant for the formation of compound A at -94’C was 685 M-‘. s-l, while a Kd at -100°C was 300 PM and an apparent EA was 9.9 kcal/mol. It



FIG. 3. Difference spectra of the intermediates in the cytochrome c oxidase-oxygen reaction. (A) Compound A, reduced oxidase (formed from “mixed-valence” state oxidase); temperature, -96°C. (B) Compound B, reduced oxidase; temperature, -65°C. (C) Compound C, reduced oxidase; temperature, -30°C. For experimental details, see Ref. 68. (Courtesy of Professor B. Chance.)

should be pointed out that this large apparent dissociation constant of oxygen from cytochrome oxidase at -100°C contrasts sharply with the Km value of 0.05-0.1 pM found at room temperature (see, for example, Refs. 77 and 78). At temperatures above -90°C compound A was converted to a so-called compound B (Fig. 3) which was accompanied by disappearance of the 590- to 591-nm maximum, shift of the 611-nm trough to 609 nm, and appearance of a broad absorption band near 780-790 nm (75). The appearance of compound B was reported to follow firstorder kinetics (K = 0.45 s-l at -78°C) and the apparent activation energy was 12.5 kcal/mol. It was further reported (75) that formation of compound B was accompanied by the appearance of a g = 2.03 epr signal. Both the epr signal and the 780- to 790-nm absorption band was attributed to oxidation of the “invisible copper,” an interpretation which, if correct, identifies these changes as the first direct measurement of the oxidation of this component.



Compound A can also be formed as the product of the oxygen reaction with cytochrome oxidase which has been reduced in the presence of CO with a subsequent excess of ferricyanide added. Chance and coworkers (75), in agreement with Greenwood et al. (72), assumed that the “invisible copper,” cytochrome CL,and the “visible” copper were all oxidized by ferricyanide prior to addition of oxygen, and thus compound A under these conditions was considered to be Cu2+a32+-02. Subsequent electron transfer at temperatures above -90°C converted compound A to compound C, whose postulated formal valency was CU’+U~~+-~Z”-. The data of data of Lindsay and co-workers (45,46) suggest, however, that in the carbon monoxide compound of cytochrome oxidase treated with ferricyanide (“mixed-valence state”) both cytochrome CQand the “invisible copper” are reduced. If this is true, compound C and the “oxygenated” oxidase of Greenwood et al. (72) could represent the formal valency CU~+-O~~--U~~+ proposed for the bound peroxide intermediate (67). Precise comparison and assignment of the various intermediates are difficult because the experiments were carried out on different materials and under nonuniform experimental conditions. Our own interpretation is as follows: Compound A with the possible formal valency state Cu+&+-O2 has the properties of an oxygen compound and would be relatively stable at low temperatures if the activation energy for the formation of the oxygen-reduced copper bond were greater than that for the oxygen-heme bond. Therefore, only when the temperature is raised to overcome this energy barrier is a bridged Cu+-02-u3’+ complex formed. With two electrons available in the immediate vicinity an internal electron transfer leads to the formation of bound peroxide, CU~+-O~~--U~~+ [compound C of Chance et al. (75); oxygenated oxidase of Greenwood et al. (72) and Orii and King (71)]. We do not include compound B as an intermediate in the scheme because the appearance of the g = 2.03 epr signal and the absorption band at 780-790 nm as well as the concomitant oxidation of cytochrome c suggest that the formation of compound B involves electron transfer



from and oxidation of the “visible” copper and cytochrome a. [This interpretation is, however, different from that given by Chance and co-workers (75, 76).] ROLE OF CYTOCHROME REGULATION OF CELLULAR


The observation that the first two sites of oxidative phosphorylation are at near equilibrium (see, for example, Refs. 79 and 80) suggests that the control of mitochondrial respiration is exerted through the cytochrome c oxidase-oxygen reaction, a step strongly displaced from equilibrium (67, 81). This means that the oxidation of reduced cytochrome c must be dependent on the mitochondrial “energy state” and that this dependence must account for the behavior of the overall respiratory rate under various conditions. Simultaneous measurement of the reduction of cytochrome c and of the respiratory rate in intact mitochondria (see Fig. 4) showed that in the presence of an uncoupler or at 10~ [ATP]/[ADP][Pi] (2 mM ADP and 10 mM Pi, no added ATP) the respiratory rate was proportional to the reduction of cytochrome c and was essentially pH independent. The respiratory rate at a given percentage reduction of cytochrome c was inhibited by the addition of ATP. The changes in the apparent rate constant corresponded to more than 95% inhibition of respiration depending on both the pH and the reduction of cytochrome c (see Fig. 4 and Ref. 67). This effect was a function of the [ATP]/ [ADP][Pi] and was uncoupler and oligomycin sensitive. A kinetic model consistent with these and other observations has been proposed (67) to provide a framework for discussion of the possible mechanism(s) of cytochrome oxidase. Since the model gives a good mathematical fit to the observed regulation of mitochondrial respiration both in suspensions of isolated mitochondria (67) and in intact cells and tissues (82), its brief description follows (Fig. 5). The active site of the reaction with oxygen is postulated to be cytochrome a3 and the “invisible copper.” The first electron entering the oxidized cytochrome oxidase from the substrate results in reduction of the “invisible copper,” which in the pres-


% Cytochrome


FIG. 4. The dependence of the mitochondrial respiratory rate on the reduction of cytochrome c at pH 7.0. The experimental points represent data obtained by suspending pigeon heart mitochondria at approximately 0.2 PM cytochrome a in a medium containing 0.2 M sucrose, 0.04 M morpholinopropane sulfonate, and 0.2 mM ethylene dinitrilotetraacetate, pH 7.0. Sodium ascorbate (5 mM), ATP, ADP, and Pi were added to stimulate respiration. The respiratory rate and the reduction of cytochrome c (550 nm minus 540 nm) were measured simultaneously and the resulting data are plotted. The added concentrations of ATP, ADP, and P, were 4 mM ATP (0); 5 mM ATP, 1 mM ADP, and 1 mM P, (0); 1 mM ATP, 1 mM ADP, and 1 mM P, (A); and 3 pg of oligomycin plus 0.023 pM S-13 (0). Control measurements with 3 pg of oligomycin plus 0.023 pM S-13 in addition to the indicated ATP, ADP, and P, were indistinguishable from those of just oligomycin and S-13. The solid curves are the simulated behavior of a suspension of mitochondria at a concentration of 0.4 pM cytochrome c (0.2 pM cytochrome a) and [ATP]/[ADP][P,] values of 10-l M-l, IO” M-’ and 4 X lo5 Me*. [From Wilson et al. (60); reprinted by permission.]



ment to form a bridged oxygen compound which is reduced in a strongly exergonic two-electron reaction to bound peroxide ( u33+-022--cu2+). The phosphorylation reactions are not considered to be kinetically limiting as near equilibrium is attained for the first two sites of oxidative phosphorylation and all three sites share common “high energy” intermediates. Fitting of the steady-state kinetic esxpression to the experimental data (67) assigns an equilibrium constant (2 x lo5 M-‘) to reaction 5 at low MtiC concentrations which is consistent with an E, value at pH 7.0 of 0.55 V for the reduction of the bound peroxide to water. The pH dependence of the equilibrium constant obtained by the fitting procedure suggests that the reaction as written consumes two or more protons (67). This model incorporates the two-electron reduction of 02 and the bound peroxide intermediate required by thermodynamic considerations as well as the role for the “invisible copper” suggested by CO binding data. The reaction site does not attain a significant level of fully reduced but unliganded form (CU+U~~+) because at the oxidation-reduction potential of greater than 0.6 V for the oxygen reaction site (70) the doubly reduced form must be at very low


j+jgFiy-y 2c3+*


‘la’* c”‘* , kOz

*TP 5


2C”*ADP*P, 41

ence of [ATP]/[ADP][Pj] near 2000 M-’ (cellular conditions) has an E, value more positive than that of cytochrome a3 (in the presence of ATP the Em~.0of cytochrome a3 is 0.155 V). Reduction of copper (reaction 1) opens the site and oxygen enters (reaction 2) in a diffusion-limited reaction (k = 1 X 10’ M-’ s-‘) forming a low affinity complex with the reduced copper. A rapid electron exchange between the Cu and the iron leads to an equilibrium mixture of two species: uz3+ Cu+-02 and CL?+ a?+-OZ. The arrival of a second electron causes reduction of the second metal atom and is accompanied by a rapid internal rearrange-








FIG. 5. A schematic representation of the reaction of the cytochrome a:~ “invisible copper” portion of cytochrome oxidase with cytochrome c and oxygen. For simplicity, no intermediate compounds are indicated which couple the redox reactions to ATP synthesis. Similarly, intermediates in the electron transport pathway between cytochrome c and the as-C” complex have been omitted. In both cases, the omitted compounds are not considered to be important at ratelimiting stages in the respiration process. [From Wilson et al. (60); reprinted by permission.]



concentrations. The reported kinetic intermediates may be tentatively related to the model as follows: Compound A (75) should be equivalent to a~‘+. 02. Cu+, an intermediate in the reaction of oxygen with the fully reduced active site in which the oxygen is bound to the iron but not to the copper. In the steady-state model at physiological temperatures this compound would not be present in any significant concentration (the condition where both cytochrome a3 and Cu are reduced does not occur to any considerable extent under respiring conditions) and is omitted from the scheme. It would occur only transiently as the second electron reaches the active site and then is immediately transferred to the oxygen. Compound B (75) because of its reported epr and optical properties is not considered to be a true intermediate in the oxygen reaction but rather the result of partial oxidation of cytochrome a and the “visible” copper. Compound C (75), the “oxygenated” oxidase of Greenwood et al. (75) and of Orii and King (71), may be equivalent to as3+. Oz2-. Cu2+ in the scheme, which can represent a real and important intermediate in the catalytic cycle. CURRENT


The controversy most central to cytochrome oxidase is that concerning the identity of cytochromes a and a3. Two different views are currently entertained in the literature. (i) Cytochromes a and a3 are two chemically distinct components of cytochrome oxidase with a priori different properties. One of the two cytochromes, cytochrome a3, has a high redox midpoint potential and reacts with added ligands, while the other, cytochrome a, has a low midpoint redox potential and is generally inactive toward ligands. However, strong interactions are present and changes in one cytochrome such as reduction or ligand binding change the properties of the other cytochrome. (ii) The two cytochromes are chemically indistinguishable but strongly interacting and reduction of, or ligand binding to, one of the cytochromes modifies the properties of the other cytochrome. The resulting behavior pattern is mathemati-



tally similar to that of two different cytochromes. Among the research groups which espouse versions of the two-cytochrome hypothesis are those associated with Beinert (32, 36,37), Chance (73-75), King (71), Palmer (41,42), Erecinska (29-31), and Wilson (38, 46, 67), whereas research groups associated with Malmstrom (84) and Van Gelder (39,83) favor the second hypothesis. Nicholls and co-workers (2, 85) and more recently Wikstrom et al. (86) prefer a “neoclassical” variant of the first hypothesis in which cytochromes a and a3 are chemically different but have similar oxidationreduction properties. Common to all of the hypotheses is the concept of heme-heme interaction which occurs between the cytochromes. This concept was originally put forward (87-89) within the framework of the “two-differentcytochromes hypothesis” and was used to interpret the observed interdependence of absorption spectra and electronic spin states of cytochromes a and a3 in the presence and absence of ligands. It was subsequently extended to generate the “twoidentical-cytochromes model” (83, 84) as well as to interpret the experimental results according to the neoclassical concepts by Nicholls and co-workers (2, 85) and by Wikstrom et al. (86). The controversy concerns primarily the extent to which the measured properties, in particular the absorption spectra and halfreduction potentials, are coupled, i.e., the strength of the interaction. The identicalcytochrome concept and the “neoclassical” hypothesis require a negative interaction energy equivalent to approximately 0.16 V in the oxidation-reduction reactions. (The first cytochrome reduced at pH 7.2 gives an E, of 0.38 V while the second gives an E, of 0.22 V.) However, if this were a straightforward coupling of the oxidation-reduction states of the cytochromes, then the E, value of the unliganded cytochrome (cytochrome a) should be on the average 0.16 V more positive when the liganded cytochrome (cytochrome ad is oxidized than when it is reduced. The experimental results presented in Table I and discussed in more detail in Refs. 90 and 91 show that it is not the case. The measured E, value of



OxidationE,” value reduction of of cytocytochrome chrome a a3

None” co CO + ATP’ NO

Reduced Reduced Reduced Reduced

0.210 0.255 0.215 0.266


Oxidized Oxidized Oxidized Oxidized

0.270 0.270 0.290 0.220






88 a 92 Unpublished results 93 go,91 90,91 go,91

a Suspensions of pigeon heart mitochontia at pH 7.2 were placed in a four-beam spectrophotometer and the absorbance changes at 552 nm minus 540 nm and at 605 nm minus 575 nm were measured simultaneously. In suspensions of anaerobic mitochondria, the measured E, of cytochromes c + cl was 225 mV (552 nm - 540 nm). The E, values for cytochrome a were measured by comparison with cytochromes c + c, in a CO-saturated medium after formation of the cytochrome as-CO compound or in an aerobic medium containing 1 mM NaCN, 506 PM Na#, or 20 mM NaN3 by titrating the reduction of the respiratory chain with trace amounts of substrate (either ascorbate plus phenaxine methosulfate or succinate at levels far below the K,,,). The resulting reduction of the cytochromes either was stepwise or progressed slowly so that in each case a log (c”/c”‘) vs log (&/a’+) was linear with a slope of 1.0. ’ Determined in normal potentiometric titrations of anaerobic suspensions of mitochondria in the presence or absence of 6 mM ATP.

cytochrome a is 0.25 + 0.04 V when cytochrome a3 is reduced and either unliganded or liganded to CO or NO and also when cytochrome a3 is oxidized in the presence of ATP (intact mitochondria) or liganded to HNa HCN, or H&3. It is clear that in areas of controversy many lines of evidence should be considered and the problem approached from all possible angles. Our understanding of the mechanism and function of cytochrome oxidase will arise from a successful union between well-designed and well-controlled experiments and innovative working hypotheses which serve as intellectual framework and guide the experimental design.

1 LEMBERG, M. R. (1963) Physiol. Rev. 49, 48-121. 2. NICHOLLS, P. AND CHANCE, B. (1974) in Molecular Mechanisms of Oxygen Activation (Hayaishi, O., eds.), pp. 479-534, Academic Press, New York. 3. CAUGHEY, W. S., WALLACE, W. T., VOLPE, T. A., AND YOSHIKAWA, S. (1976) in The Enzymes (Boyer, P. D., ed.), Vol. 13, pp. 299-337, Academic Press, New York. 4. POYTON, R. O., AND SCHATZ, G. (1975) J. Bill. Chem. 250,752-761. 5. SEBALD, W., MACHLEIDT, W., AND OTTO, J. (1973) Eur. J. Biochem. 38,311-324. 6. BRIGGS, M., KAMP, P. F., ROBINSON, N. C., AND CAPALDI, R. A. (1975) Biochemistry 15, 5123-5128. 7. PHAN, S. H., AND MAHLER, H. R. (1976) J. Biol. Chem. 251,257-263. 8. DOWNER, N. W., ROBINSON, N. C., AND CAPALDI, R. A. (1976) Biochemistry 15.2930-2936. Chem. 236, 9. YONETANI, T. (1961) J. Biol. 1680-1686. 10. SUN, F. F., PREZBINDOWSKI, K. S., CRANE, F. L., AND JACOBS, E. E. (1968) Biochim. Biophys. Acta 153,804-818. 11. CAPALDI, R. A., AND HAYASHI, H. (1972) FEBS Lett. 26, 261-263. 12. CAPALDI, R. A., BELL, R. L., AND BRANCHEK, T. (1977) B&hem. Biaphys. Res. Commun. 74, 425-433. 13. EYTAN, G. D., AND SCHATZ, G. (1975) J. Biol. Chem. 250, 767-774. 14. EYTAN, G. D., CARROLL, R. C., SCHATZ, G., AND RACKER, E. (1975) J. Biol. Chem. 250, 8598-8603. 15. CARROLL, R., AND EYTAN, G. D. (1975) Fed. Proc. 34,2036. 16. ERECI~SKA, M. (1977) Biochem. Biophys. Res. Commun. 76.495-501. 17. BRIGGS, M. M., AND CAPALDI, R. A. (1977) Biochemistry 16.73-77. 18. POYTON, R. O., AND SCHATZ, G. (1975) J. Bi& Chem. 250,762-766. 19. PHAN, S. H., AND MAHLER, H. R. (1976) J. Biol. Chem. 251.270-276. 20. KOMAI, H., AND CAPALDI, R. A. (1973) FEBS Lett. 30.273-276. 21. YAMAMOTO, T., AND ORII, Y. (1974) J. Biochem. 75, 1081-1089. 22. Yu, C. A. Yu, L., AND KING, T. E. (1977) Biochem. Biophys. Res. Common. 74,670-676. 23. RUBEN, G. C., TELFORD, J. N., AND CARROLL, R. C. (1976) J. Cell Biol. 68, 724-739. 24. HACKENBROCK, C. R., AND HAMMON, K. M. (1975) J. Biol. Chem. 250,9185-9197. 25. JUNGE, W., AND DEVAULT, D. (1975) Biochim. Biophys. Acta 408,200-214.



26. HENDERSON, R., CAPALDI, R. A., AND LEIGH, J. S. (1977) J. Mol. Biol. 112, 631-648. 27. BLASIE, J. K., ERECI~SKA, M., SAMUELS, S., AND LEIGH, J. S., JR. (1977) Biophys. J. 17,63a. 28. BLASIE, J. K., ERECII;JSKA, M., SAMUELS, S., AND LEIGH, J. S., JR. (1978) Biochim. Biophys. Acta 501,33-52. 29. ERECII%KA, M., WILSON, D. F., AND BLASIE, J. K. (1978) Biochim. Biophys. Acta 501.53-62. 30. ERECI%KA, M., WILSON, D. F., AND BLASIE, J. K. (1977) FEBS Lett. 76, 235-239. 31. ERIXINSKA, M., WILSON, D. F., AND BLASIE, J. K. (1978) Biochim. Biophys. Acta 501.63-71. 32. HAHTZELJ., C. R., HANSEN, R. E., AND BEINERT, H. (1973) Proc. Nat. Acad. Sci. USA 70, 2477-2481. 33. LEIGH, J. S., JIM., WIISON, D. F., OWEN, C. S., AND KING, T. E. (1974) Arch. Biochem. Biophys. 160,476-486. 34. WILSON, D. F., ERECIIQSKA, M., LINDSAY, J. G., LEIGH, J. S., Jn., AND OWEN, C. S. (1975) in Mitochondria/Biomembranes, Proceedings of the 10th FEBS Meeting, pp. 195-210. 35. WILSON, D. F., ERECIIQSKA, M., AND OWEN, C. S. (1975) Arch. Biochem. Biophys. 175, 160-172. 36. BEINERT, H., HANSEN, R. E., AND HARTZELL, C. R. (1976) Biochim. Biophys. Acta 423.339-355. 37. HARTZELL, C. R., AND BEINERT, H. (1976) Biochim. Biophys. Acta 423,323-338. 38. WILSON, D. F., ERECIIQSKA, M., AND BROCKI.E. EIIJHST, E. S. (1972) Arch. Biochem. Biophys. 151, 180-187. 39. VAN BLJIJHF,N, K. J. H., NICHOLLS, P., AND VAN GF.LDER, B. V. (1972) B&him. Biophys. Acta 256, 258-276. 40. BWITTAIN, T., SPRINGALL, T., GREENWOOD, C., AND THOMSON, A. T. (1976) Biochem. J. 159, 811-813. 41. PALMER, G., BABCOCK, G. T., AND VICKERY, L. E. (1976) Proc. Nat. Acad. Sci. USA 73, 2206-2210. 42. BARCOCK, G. T., VICKERY, L. E., AND PAI,MER, G. (1976) J. Biol. Chem. 254, 7907-7919. 43. FAI,K, K.-E., VANNGARD, T., AND ANGSTROM, T. (1977) FEBS Lett. 75, 23-27. 44. Hr:, V. W., CHAN, S. I., AND BROWN, G. S. (1977) Proc. Nat. Acad. Sci. USA 74, 3821-3825. 45. LINDSAY, J. G.. AND WILSON, D. F. (1974) FEBS Lett. 48, 45-49. 46. LINDSAY, J. G., OWEN, C. S., AND WILSON, D. F. (1975) Arch. Biochem. Biophys. 169, 492-505. 47. ANDRRSON, J. L., KIJWANA, T., AND HARTZELL, C. R. (1976) Biochemistry 15,3847-3855. 48. WIISON, D. F., AND MIYATA, Y. (1977) Biochim. Biophys. Acta 461, 218-230. 49. WEVEH, R., VAN DHOOGE, J. H., MUIJSERS, A. O., BAKKER, E. P., AND VAN GELDER, B. F. (1977) Eur. J. Biochem. 73, 149-154. 50. TZAOOI,OFF, .4., AND WHARTON, D. C. (1965) J.



Biol. Chem. 240,2628-2633. 51. GREENWOOD, C., WILSON, M. T., AND BRUNORI, M. (1974) Biochem. J. 137, 205-215. 52. NICHOLLS, P., PETERSEN, L. C., MILLER, M., AND HANSEN, F. B. (1976) Biochim. Biophys. Acta 449, m-196. 53. NICHOLLS, P. (1975) Biochim. Biophys. Acta 398, 24-35. 54. WEVER, R., VAN GELDER, B. F., AND DERVARTANIAN, D. V. (1975) Biochim. Biophys. Acta 387, 189-193. 55. NICHOLLS, P. (1975) Biochem. Biophys. Res. Comman. 67, 610-616. 56. NICHOLLS, P. (1976) B&him. Biophys. Acta 430, 13-29. 57. MITCHELL, P. (1966) Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research, Bodmin, England. 58. DRACHEV, L. A., JASAITIS, A. A., KAULEN, A. D., KONDRASHIN, A. A., CHU, L. V., SEMENOV, A. Y., SEVERINA, I. I., AND SKULACHEV, V. P. (1976) J. Biol. Chem. 254, 7072-7076. 59. WIKSTROM, M. K. F. (1977) Nature (London) 266, 271-273. 60. WILSON, M. T., GREENWOOD, C., BHUNORI, M., AND ANTONINI, E. (1975) Biochem. J. 147, 145-153. 61. ANDREASSON, L.-E. (1975) Ear. J. Biochem. 53, 591-597. 62. GREENWOOD, C., BRITTAIN, T., WILSON, M., ANI> BRIJNORI, M. (1976) Biochem. J. 157, 591-598. 63. ANTONINI, E., BR~JNORI, M., COI,OSIMO, A., GREENWOOD, C., AND WIISON, M. T. (1977) Proc. Nat. Acad. Sci. USA 74,3128-3132. 64. EHREDE, B., HAIGHT, G. P., AND KAMEN, M. D. (1976) Proc. Acad. Sci. USA 73, 113-117. 65. FERGUSON-MILLER, S., BRAUTIGAN, D. L., ANI) MARGOLIASH, E. (1976) J. Biol. Chem. 254, 1104-1115. 66. ERECI$X+KA, M. (1975) Arch. Biochem. Biophys. 169, 199-208. 67. WILSON, D. F., OWEN, C. S., AND HOI.IAN, A. (1977) Arch. Biochem. Biophys. 182, 749-762. 68. GEORGE, P. (1965) in Oxidases and Related Redox Systems (King, T. E., Mason, H. S., and Morrison, M., eds.), Vol. 1, pp. 3-33, Wiley, New York. 69. WOOD, P. M. (1975) FEBS Lett. 44, 22-24. 70. WIIZON, D. F., ERECI~SKA, M., AND DUTTON, P. L. (1974) Anna. Rev. Biophys. Bioeng. 3, 203-230. 71. 0~11, Y., AND KING, T. E. (1972) FEBS Lett. 21, 199-202. 72. GREENWOOD, C., WILSON, M. T., AND BHUNORI, M. (1974) Biochem. J. 137,202-215. 73. CHANCE, B., ERECI~~SKA, M., AND CHANCE, E. M. (1973) in Oxidases and Related Redox Systems II (King, T. E., Mason, H. S., and Morrison, M., eds.), pp. 851-866, University Park Press, Baltimore.



74. CHANCE, B., SARONIO, C., AND LEIGH, J. S., JR. (1975) Proc. Natl. Acad. Sci. USA 72, 1635-1640. 75. CHANCE, B., SARONIO, C., AND LEIGH, J. S., JR. (1975) J. Biol. Chem. 250,9226-9237. 76. CHANCE, B., AND INGLEDEW, J. (1977) Fed. Proc. 36, 727. 77. OSHINO, N., SUGANO, T., OSHINO, R., AND CHANCE, B. (1974) Biochim. Biophys. Acta 368, 298-310. 78. PETERSEN, L. C., NICHOLLS, P., AND DEGN, H. (1974) Biochem. J. 142,247-252. 79. ERECI&SKA, M., VEECH, R. L., AND WILSON, D. F. (1974) Arch. Biochem. Biophys. 160, 412-421. 80. WILSON, D. F., STUBBS, M., OSHINO, N., AND ERECI~SKA, M. (1974) Biochemistry 13, 5305-5311. 81. OWEN, C. S., AND WILSON, D. F. (1974) Arch. Biochem. Biophys. 161,581-591. 82. ERECI~SKA, M., NISHIKI, K., AND WILSON, D. F. (1978) Amer. J. Physiol., in press. 83. TIESJEMA, R. H., MUIJSERS, A. O., AND VAN GELDER, B. F. (1973) Biochin. Biophys. Acta 305, 19-28.



84. MALMSTROM, B. (1974) Quart. Rev. Biophys.



85. NICHOIU, P., AND PETERSEN, L. C. (1974) Biochim. Biophys. Acta 357.462-467. 86. WIKSTROM, M. K. F., HARMON, H. J., INGLEDEW, W. J., AND CHANCE, B. (1976) FEBS Lett. 65, 259-277. 87. VAN GELDF,R, B. F., AND BEINERT, H. (1969) Biochim. Biophys. Acta 189, l-24. 88. WILSON, D. F., LINDSAY, J. G., AND BROCKLEHURST, E. S. (1972) Biochin. Biophys. Acta 256,277-286. 89. WILSON, D. F., AND LEIGH, J. S., JR. (1972) Arch. Biochem. Biophys. 150.154-163. 90. WILSON, D. F., DUTTON, P. L., ERECI~SKA, M., AND LINDSAY, J. G. (1973) in. Mechanisms in Bioenergetics (Azzone, G. F., Ernster, L., Papa, S., QuaglarieIIo, E., and SiIiprandi, N., eds.), pp. 527-533, Academic Press, New York. 91. WILSON, D. F., AND LEIGH, J. S., JR. (1974) Ann. N. Y. Acad. Sci. 227,630-635. 92. WILSON, D. F., AND BROCKLEHURST, E. S. (1973) Arch. Biochem. Biophys. 158,200-212.

Cytochrome c oxidase: a synopsis.

ARCHIVES OF BIOCHEMISTHY AND BIOPHYSICS Vol. 188, No 1, May, pp. I-14, 1978 Invited Cytochrome MARIA Department of Biochemistry c Oxidase: ERECINS...
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