Reaction of formate with the fast form of cytochrome oxidase: a model for the fast to slow conversion.

The ability to isolate preparations of cytochrome oxidase which are highly homogeneous has facilitated a study of the effects of various reagents on the purified enzyme. The addition of either sodium formate, formamide, formaldehyde, or sodium nitrite to enzyme which reacts in a single rapid kinetic phase with cyanide causes a blue-shift of 4-6 nm of the net (cytochrome a + cytochrome a3) Soret maximum. Only the derivative prepared by adding sodium formate demonstrates measurable intensity in the g' = 12 region of the low-temperature electron paramagnetic resonance (EPR) spectrum. This g' = 12 resonance is characteristic of cytochrome oxidase which has undergone a modification at the binuclear center and thereby reacts sluggishly with cyanide. As the site of cyanide binding in resting enzyme as been demonstrated to be CuB [Yoshikawa, S., & Caughey, W.S. (1990) J. Biol. Chem. 265, 7945-7958], it is proposed that formate can bind to CuB and the fast to slow transition is rationalized by using this proposal. The g' = 12 signal is also produced upon the addition of sodium formate to mitochondrial preparations, suggesting that the species responsible for this behavior may have possible physiological relevance. Physical properties of the formate derivative and data for other reagents reacted with the fast-reacting enzyme preparation are presented.     

"Peroxidatic" form of cytochrome oxidase as studied by X-ray absorption spectroscopy

X-ray absorption spectroscopy shows pulsed oxidase to be similar to resting oxidase but to lack the sulfur bridge between iron and copper of active sites (Powers, L., Y. Ching, B. Chance, and B. Muhoberac, 1982, Biophys. J., 37[2, Pt. 2]: 403a. [Abstr.] ) The first shell ligands and bond lengths of the pulsed oxidase active site heme most clearly fit the ferric peroxidases from horseradish and yeast, and the pulsed oxidase cyanide compound resembles the low spin hemoprotein cyanide compounds. The structural results are consistent with an aquo or a peroxo form for pulsed oxidase as is also observed by optical studies. These structural and chemical data are consistent with a role for the pulsed forms in a cyclic peroxidatic side reaction in which the pulsed and pulsed peroxide compounds act as peroxide scavengers. The peroxidatic role of cytochrome oxidase in the nonsulfur bridged form suggests the renaming of the "oxygenated" or "pulsed" forms on a functional basis as "peroxidatic" forms of cytochrome oxidase.     

Binding of isotopically labeled substrates, inhibitors, and cyanide by protocatechuate 3,4-dioxygenase

Binding of ligands to the active site Fe3+ of protocatechuate 3,4-dioxygenase is investigated using EPR-detected transferred hyperfine coupling from isotopically labeled substrates, inhibitors, and cyanide. Broadening is observed in EPR resonances from the anaerobic enzyme complex with homoprotocatechuate (3,4-dihydroxyphenylacetate), a slow substrate, enriched with 17O (I = 5/2) in either the 3-OH or the 4-OH group. This shows that this substrate binds directly to the Fe3+ and strongly suggests that an iron chelate can be formed. Cyanide is known to bind to the enzyme in at least two steps, forming first a high spin and then a low spin complex (Whittaker, J. W., and Lipscomb, J. D. (1984) J. Biol. Chem. 259, 4487-4495). Hyperfine broadening from [13C]cyanide (I = 1/2) is observed in the EPR spectra of both complexes, showing that cyanide is an Fe3+ ligand in each case. Cyanide binding is also at least biphasic in the presence of protocatechuate (PCA). The initial high spin enzyme-PCA-cyanide complex forms rapidly and exhibits a unique EPR spectrum. Broadening from PCA enriched with 17O in either the 3-OH or the 4-OH group is detected showing that PCA binds to the iron, probably as a chelate complex. In contrast, no broadening from [13C]cyanide is detected for this complex suggesting that cyanide binds at a site away from the Fe3+. Steady state kinetic measurements of cyanide inhibition of PCA turnover are consistent with two rapidly exchanging cyanide binding sites that inhibit PCA binding and which can be simultaneously occupied. Formation of the nearly irreversible, low spin enzyme-PCA-cyanide complex is competitively inhibited by PCA. Transient kinetics of the formation of this complex are second order in cyanide implying that two cyanides bind. Broadening in the EPR spectrum of this complex is detected from [13C]cyanide, but not from [17O]PCA, suggesting that PCA is displaced. This study provides the first direct evidence for chelation of the active site Fe3+ by substrates and for a small molecule binding site away from the iron in intradiol dioxygenases.     

Effect of cyanide binding on the copper sites of cytochrome c oxidase: an X-ray absorption spectroscopic study

Cu x-ray absorption spectroscopy (XAS) has been used to investigate the effect of cyanide treatment on the structures of the copper sites in beef heart cytochrome c oxidase. The Cu K-edge spectrum changes significantly upon cyanide binding to resting state enzyme, as does the Cu extended x-ray absorption fine structure (EXAFS) spectrum. The Cu EXAFS Fourier transfer (FT) exhibits an enhanced peak for the cyanide-treated enzyme in the region containing the Cu...Fe peak in the resting state FT (at R' approximately equal to 2.6-2.7 A). This peak in the cyanide-treated sample is hypothesized to arise from "outer shell" scattering from a linear Cu-cyanide moiety, suggesting cyanide binding to CuB only (CuB 2+-CN-) or cyanide bridging between the Fe of heme a3 and CuB (Fe3+-(CN-)-CuB 2+).     

Interrelationships of copper, cytochrome oxidase, phospholipid synthesis and adenine nucleotide binding.

Studies are reported on the interrelationships in liver mitochondria of copper status, cytochrome oxidase activity, adenine nucleotide binding capacity and phospholipid synthesis. Direct exposure of mitochondria to cyanide or diethyldithiocarbamate depressed cytochrome oxidase activity; ADP-binding and phospholipid synthesis. Fractionation of mitochondria to increase the specific activity of cytochrome oxidase about 10-fold did not increase the affinity to bind ADP. Ageing of mitochondria or dialysis of mitochondria or mitochondrial membrane preparations against water or diethyldithiocarbamate at 0--2 degrees for 18 h did not decrease cytochrome oxidase activity or copper content of reisolated and resuspended mitochondria or mitochondrial membrane preparations, but considerably reduced the affinity to bind ADP. The respiratory inhibitors, fluoride and azide, at concentrations inhibitory to cytochrome oxidase did not reduce ADP-binding or phospholipid synthesis. Atractyloside did not inhibit cytochrome oxidase activity but did inhibit ADP-binding and phospholipid synthesis. Pre-incubation of mitochondrial membrane preparations with Cu++ increased the copper content and ADP-binding affinity. The results indicate that cytochrome oxidase is not the ADP-binding site of the mitochondrial membrane system and that reduced cytochrome oxidase activity per se does not depress binding affinity. Copper appears to be a component of the adenine nucleotide binding sites of mitochondrial membranes because the copper-complexing agents, cyanide and diethyldithiocarbamate, depressed ADP-binding, while increased mitochondrial membrane copper content increased ADP-binding.     

Reduction of cytochrome oxidase by 5,10-dihydro-5-methylphenazine: kinetic parameters from rapid-scanning stopped-flow experiments

The kinetics of the reduction of resting cytochrome oxidase and of its cyanide complex by 5,10-dihydro-5- methylphenazine (MPH) have been characterized by rapid-scan and fixed-wavelength stopped-flow spectrophotometry in the Soret, visible, and near-IR spectral regions. In this study, we focused on a form of the resting enzyme that is characterized by a Soret absorption maximum at 424 nm. These experiments complement earlier work on the reduction of a 418 nm absorbing form of the resting enzyme [ Halaka , F.G., Babcock , G. T., & Dye, J. L. (1981) J. Biol. Chem. 256, 1084-1087]. The reduction of cytochrome a is accomplished in a second-order reaction with a rate constant of 3 X 10(5) M-1 s-1. The reduction of the 830-nm absorber, Cua, is closely coupled to but lags the reduction of cytochrome a; we have resolved a rate constant of about 20 s-1 for the copper reduction. The reduction of cytochrome a proceeds with a rate constant that is nearly independent of the spectral properties of the resting enzyme and of the ligation state of cytochrome a3. The reduction of cytochrome a3 occurs by slow, intramolecular electron transfer. We have resolved two phases for this process that have rate constants of approximately 0.2 s-1 and approximately 0.02 s-1 for both the 418- and 424-nm forms of the resting enzyme. It appears, therefore, that spectroscopic heterogeneity at the cytochrome a3 site in the resting enzyme exerts very little influence on the kinetics of the anaerobic reduction of the oxidase metal centers. From this we conclude that the rate of electron transfer to the a3 site is probably controlled by the protein conformation and not primarily by local factors within the a3 environment.     

Kinetic studies on the reaction between cytochrome c oxidase and ferrocytochrome c.

In stopped-flow experiments in which oxidized cytochrome c oxidase was mixed with ferrocytochrome c in the presence of a range of oxygen concentrations and in the absence and presence of cyanide, a fast phase, reflecting a rapid approach to an equilibrium, was observed. Within this phase, one or two molecules of ferrocytochrome were oxidized per haem group of cytochrome a, depending on the concentration of ferrocytochrome c used. The reasons for this are discussed in terms of a mechanism in which all electrons enter through cytochrome a, which, in turn, is in rapid equilibrium with a second site, identified with 'visible' copper (830 nm-absorbing) Cud (Beinert et al., 1971). The value of the bimolecular rate constant for the reaction between cytochromes c2+ and a3+ was between 10(6) and 10(7) M(-1)-S(-1); some variability from preparation to preparation was observed. At high ferrocytochrome c concentrations, the initial reaction of cytochrome c2+ with cytochrome a3+ could be isolated from the reaction involving the 'visible' copper and the stoicheiometry was found to approach one molecule of cytochrome c2+ oxidized for each molecule of cytochrome a3+ reduced. At low ferrocytochrome c concentrations, however, both sites (i.e. cytochrome a and Cud) were reduced simultaneously and the stoicheiometry of the initial reaction was closer to two molecules of cytochrome c2+ oxidized per molecule of cytochrome a reduced. The bleaching of the 830 nm band lagged behind or was simultaneous with the formation of the 605 nm band and does not depend on the cytochrome c concentration, whereas the extinction at the steady-state does. The time-course of the return of the 830 nm-absorbing species is much faster than the bleaching of the 605 nm-absorbing component, and parallels that of the turnover phase of cytochrome c2+ oxidation. Additions of cyanide to the oxidase preparations had no effect on the observed stoicheiometry or kinetics of the reduction of cytochrome a and 'visible' copper, but inhibited electron transfer to the other two sites, cytochrome a3 and the undetectable copper, Cuu.     

Resonance Raman evidence for low-spin Fe2+ heme a3 in energized cytochrome c oxidase: implications for the inhibition of O2 reduction

Resonance Raman (RR) spectra are reported for reduced submitochondrial particles (SMP) with excitation at 441.6 nm, where Raman bands of the cytochrome c oxidase heme a groups are selectively enhanced. Addition of ATP to energize the membranes induces the formation of a new band at 1644 cm-1 and partial loss of intensity in a band at 1567 cm-1. These changes are modeled by adding cyanide to reduced cytochrome c oxidase and are attributed to partial conversion of cytochrome (cyt) a3 from a high-spin to a low-spin state. This conversion is abolished by addition of excess oligomycin, an ATPase inhibitor, or FCCP, an uncoupler of proton translocation, and is reversed when the ATP is consumed. The observed spin-state conversion is attributed to the binding of an endogenous ligand to the cyt a3 Fe atom. This ligation is suggested to be induced by a local increase in pH and/or by a global conformation change associated with the generation of a transmembrane potential. Since O2 binding requires a vacant coordination site at cyt a3, the ligation of this site must retard O2 reduction and could thus provide a simple mechanism for energy-linked regulation of respiration. No changes in the RR spectrum were observed upon adding Ca2+ or H+ to reduced cytochrome c oxidase. The cyt a3 spin-state change associated with membrane energization is unrelated to the cyt a absorption red shift induced by adding Ca2+ or H+ to cytochrome c oxidase.     

The interactions of cytochrome c and porphyrin cytochrome c with cytochrome c oxidase. The resting, reduced and pulsed enzymes

Cytochrome c oxidase forms tight binding complexes with the cytochrome c analog, porphyrin cytochrome c. The behaviour of the reduced and pulsed forms of the oxidase with porphyrin cytochrome c have been followed as functions of ionic strength; this behaviour has been compared with that of the resting oxidase [Kornblatt, Hui Bon Hoa and English (1984) Biochemistry 23, 5906-5911]. All forms of the cytochrome oxidase studied bind one porphyrin cytochrome c per 'functional' cytochrome oxidase (two heme a); it appears as though porphyrin cytochrome c and cytochrome c compete for the same site on the oxidase. The resting enzyme binds cytochrome c 8 times more strongly than porphyrin cytochrome c; the reduced enzyme, in contrast, binds the two with almost equal affinity. In all three cases, resting, pulsed and reduced, the heme-to-porphyrin distance is estimated to be about 3 nm. The tight-binding complexes formed between cytochrome oxidase and porphyrin cytochrome c can be dissociated by salt. Debye-Hückel analysis of salt titrations indicate that the resting enzyme and the reduced enzyme are similar in that the product of the interaction charges on the two proteins is about -14. The product of the charges for the pulsed enzyme is -25, indicating that on average another positive and negative charge take part in the interaction of the two proteins. While there is one tight binding site for cytochrome c per two heme a, cytochrome c is able to 'communicate' with four heme a. In the absence of cytochrome c, electron transfer from tetramethylphenylenediamine to the oxidase to oxygen results in the conversion of the resting form to the 'oxygenated'; in the presence of cytochrome c, the same electron transfer results in the appearance of the 'pulsed' form. Cytochrome c titrations of the enzyme show that a ratio of only one cytochrome c to four heme a is sufficient to convert all the oxidase to the 'pulsed' form. Porphyrin cytochrome c, like cytochrome c, catalyzes the same conversion with the same stoichiometry. The binding data and salt effects indicate that major structural alterations occur in the oxidase as it is converted from the resting to the partially reduced and subsequently to the pulsed form.     

A re-examination of the reactions of cyanide with cytochrome c oxidase

Experiments were performed to examine the cyanide-binding properties of resting and pulsed cytochrome c oxidase in both their stable and transient turnover states. Inhibition of the oxidation of ferrocytochrome c was monitored as a function of cyanide concentration. Cyanide binding to partially reduced forms produced by mixing cytochrome c oxidase with sodium dithionite was also examined. A model is presented that accounts fully for cyanide inhibition of the enzyme, the essential feature of which is the rapid, tight, binding of cyanide to transient, partially reduced, forms of the enzyme populated during turnover. Computer fitting of the experimentally obtained data to the kinetic predictions given by this model indicate that the cyanide-sensitive form of the enzyme binds the ligand with combination constants in excess of 10(6) M-1 X s-1 and with KD values of 50 nM or less. Kinetic difference spectra indicate that cyanide binds to oxidized cytochrome a33+ and that this occurs rapidly only when cytochrome a and CuA are reduced.     

Cyanide inhibition of cytochrome c oxidase. A rapid-freeze e.p.r. investigation.

The inhibition of cytochrome c oxidase by cyanide, starting either with the resting or the pulsed enzyme, was studied by rapid-freeze quenching followed by quantitative e.p.r. It is found that a partial reduction of cytochrome oxidase by transfer of 2 electron equivalents from ferrocytochrome c to cytochrome a and CuA will induce a transition from a closed to an open enzyme conformation, rendering the cytochrome a3-CuB site accessible for cyanide binding, possibly as a bridging ligand. A heterogeneity in the enzyme is observed in that an e.p.r. signal from the cytochrome a3 3+-HCN complex is only found in 20% of the molecules, whereas the remaining cyanide-bound a3-CuB sites are e.p.r.-silent.     

Redox-cycled oxidase. One of the reaction products of reduced cytochrome c, cytochrome c oxidase, and dioxygen

Pulsed and oxygenated forms of cytochrome c oxidase are believed to be variants of the oxidized enzyme. They were produced as a consequence of one or more reduction-oxidation cycles of the resting form and are characterized by an increase of the alpha band intensity and a red-shift of the Soret absorption band to 428 nm. The rate of decay of these species back to the resting enzyme varies appreciably and appears to depend on the nature of the reductant and/or oxidant used in their preparation. Here we report that if resting oxidase is incubated with either reduced or oxidized cytochrome c and then exposed to dioxygen, an activated form is rapidly produced which appears to be more oxidized than the starting material. This finding suggest some degree of partial reduction of the resting enzyme, but this by itself cannot explain the extent of activation. Our results further question the significance of the optical spectral "signature" of the oxygenated (Okunuki, K., and Sekuzu, I. (1954) Seitaino Kagaka 5, 265-272), pulsed (Antonini, E., Brunori, M., Colosimo, A., Greenwood, C., and Wilson, M. T. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3128-3132), and "420 nm" species (Kumar, C., Naqui, A., and Chance, B. (1984) J. Biol. Chem. 259, 2073-2076, 11668-11671), which are thought to be activated forms of oxidized cytochrome c oxidase.     

Kinetic studies on the binding of cyanide to oxygenated cytochrome c oxidase.

The reaction of cyanide with oxygenated cytochrome c oxidase was followed by means of flow-flash techniques. The oxygenated form, produced after photolysis of the partially reduced CO complex in the presence of cyanide and O2, shows cyanide-binding properties distinct from those of both the oxidized and the reduced forms of the protein. The binding is a single process (k = 22M-1-S-1) linearly dependent on cyanide concentration to as high as 75 mM. It is suggested that the oxygenated form is a conformational variant of the oxidized protein.     

Characterisation of 'fast' and 'slow' forms of bovine heart cytochrome-c oxidase.

We have prepared cytochrome-c oxidase from bovine heart (using a modification of the method of Kuboyama et al. (1972) J. Biol. Chem. 247, 6375-6383) which binds cyanide rapidly, shows no kinetic distinction between the two haems on reduction by dithionite, has a Soret absorption maximum above 424 nm, and has a negligible 'g' = 12' EPR signal. On incubation at pH 6.5 this 'fast' oxidase reverts to the 'slow' ('resting') form characterised by slow cyanide binding, slow reduction of haem a3 by dithionite, a blue-shifted Soret maximum and a large 'g' = 12' signal. Incubation of 'fast' oxidase with formate produces a form of the enzyme with properties almost identical to those of 'slow' oxidase. The kinetics of formate binding to 'fast' oxidase are found to be biphasic, revealing the presence of at least two 'fast' subpopulations in our preparations. Evidence is presented that there is an equilibrium mixture of high-spin and low-spin forms of haem a3 in both 'fast' subpopulations at room temperature. Incubation of 'fast' oxidase with chloride or bromide at pH 6.5 produces forms of oxidase with much lower rates of cyanide binding. Our working hypothesis is that formate mimics a binuclear centre ligand which is present in the 'slow' form of cytochrome oxidase. Although we show that chloride and bromide can also be ligands of the binuclear centre, possibly onto CuB, we can rule out either of these being the ligand present in the 'slow' enzyme. We will argue that the 'fast' and 'slow' forms of oxidase are equivalent to the 'pulsed' and 'resting' forms of oxidase, respectively.     

The conformational states of cytochrome oxidase in the mitochondrion.

THE Soret spectrum of "resting" cytochrome oxidase in cytochrome-c depleted mitochondria has been determined. The spectrum obtained is dependent on the rate at which the oxidase is turning over. In the least active preparations, the spectrum is almost pure "oxidized" oxidase. With increasing activity the spectrum is converted to a mixture of "oxidized" and "oxygenated" oxidases. It is concluded that the same conformational differences between the two non-reduced forms that are found in the purified enzyme also occur in these cytochrome-c depleted mitochondria.     

Use of spin-labelled substrate analogs for a comparative study of microsomal cytochrome P-450 and P-448

The previously described, iodine-labeled alkylating stable nitroxyl radicals located at different distances between the N-O. group and the iodine atom were used for a comparative study of the structure of microsomal cytochromes P-450 and P-448 active centers. The radicals were shown to change the optical spectra of Fe3+ located in the active site of the enzyme that are similar to those induced by cytochrome P-450 substrates. Some differences in the type of the radicals binding to control, phenobarbital- and 3-methylcholanthrene-induced microsomes were revealed. The alkylating radical substrate analogs covalently bound to microsomal cytochrome P-450 in the vicinity of the active center, resulting in the inhibition of oxidation of type I and II substrates (e. g., aniline and naphthalene). The value of the spectral binding constant (Ks) for naphthalene in the presence of the radical covalently bound to the cytochrome P-450 active center showed a tendency to increase. Using the ESR technique, the interaction between Fe3+ and the radical localized in the active site of cytochrome P-450 was demonstrated. The contribution of Fe3+ to the relaxation of the radicals covalently bound to cytochrome P-450 was evaluated from the values of the spin label ESR spectra saturation curves at 77K. The distances between the N-O. group of these radicals and Fe3+ in the enzyme active center for the three types of microsomes were determined. The data obtained point to structural peculiarities of the active center of cytochrome P-450, depending on the microsomal type.     

Purification of cytochrome-c oxidase retaining its pulsed form

A new purification procedure for cytochrome-c oxidase from bovine heart mitochondria is described. The enzyme was purified by selective solubilization in Triton X-100 and subsequent hydroxyapatite and gel chromatography. The preparation was highly pure and active. The subunit composition and steady-state kinetics were found to be the same as those reported for other preparations. In contrast to most of the previously published protocols the method presented here resulted in a preparation which had a rapid intramolecular electron transfer from cytochrome a to cytochrome a3, i.e. it was found to have retained its pulsed state. This correlated with monoexponential cyanide-binding kinetics. The formation of resting kinetics and biphasic cyanide-binding kinetics was shown to be induced by a short incubation at pH 5.0.     

Effects of cholesterol analogues and inhibitors on the heme moiety of cytochrome P-450scc: a resonance Raman study

Interactions of cholesterol analogues and inhibitors with the heme moiety of cytochrome P-450scc were examined by resonance Raman spectroscopy. The Raman spectra of ferric cytochrome P-450scc complexed with inhibitors such as cyanide, phenyl isocyanide, aminoglutethimide, and metyrapone were characteristic of low-spin state and were very similar. However, the effect of exchange of the sixth ligand from the oxygen atom (ferric low-spin state) to the nitrogen atom upon aminoglutethimide and metyrapone binding was seen as down-frequency shifts of the v3 band from 1503 to 1501 and 1502 cm-1, respectively, while cyanide and phenyl isocyanide binding caused an up-frequency shift of the v3 band to 1505 cm-1. The effects of cholesterol analogues [22(R)-hydroxycholesterol, 22(S)-hydroxycholesterol, 22-ketocholesterol, 20(S)-hydroxycholesterol, and 25-hydroxycholesterol] on a Fe2+-CO stretching frequency of cytochrome P-450scc in ferrous CO form were examined. The 22(R)-hydroxycholesterol complex could not give a clear Fe2+-CO stretching Raman band due to a strong photodissociability. 22(S)-Hydroxycholesterol and 25-hydroxycholesterol complexes gave the Raman bands at 487 and 483 cm-1, respectively, whereas 20(S)-hydroxycholesterol and 22-ketocholesterol complexes gave Fe2+-CO stretching frequencies (478 cm-1) almost identical with that without substrate (477 cm-1). These findings suggest the existence of the following physiologically important natures of the cytochrome P-450scc active site: (1) there is a strong steric interaction between heme-bound carbon monoxide and the 22(R)-hydroxyl group or the 22(R)-hydrogen of the steroid side chain and (2) the hydroxylation at the 20S position may cause a conformational change of the side-chain group relative to the heme.     

Two-electron reduction is required for rapid internal electron transfer in resting, pulsed and oxygenated cytochrome c oxidase

The resting as well as the 420 nm and 428 nm forms of cytochrome oxidase have been studied in kinetic experiments with an excess of enzyme over reduced cytochrome c. No difference was found in the behavior of the two activated forms. With all three forms, a fraction of cytochrome a was reoxidized with a rate which was much lower than kcat. This suggests that intramolecular transfer to the dioxygen-reducing site occurs only if both cytochrome a and CuA are reduced. An initial rapid phase in the oxidation of cytochrome a in the pulsed and oxygenated enzymes is related to the presence of a three-electron-reduced dioxygen intermediate. The increased catalytic activity of pulsed and oxygenated oxidase can be explained on the basis of a shift in the redox equilibrium between cytochrome a and CuA.     

ATP induces conformational changes in mitochondrial cytochrome c oxidase. Effect on the cytochrome c binding site

ATP influences the kinetics of electron transfer from cytochrome c to mitochondrial oxidase both in the membrane-embedded and detergent-solubilized forms of the enzyme. The most relevant effect is on the so-called "high affinity" binding site for cytochrome c which can be converted to "low affinity" by millimolar concentrations of ATP (Ferguson-Miller, S., Brautigan, D. L., and Margoliash, E. (1976) J. Biol. Chem. 251, 1104-1115). This phenomenon is characterized at the molecular level by the following features. ATP triggers a conformational change on the water-exposed surface of cytochrome c oxidase; in this process, carboxyl groups forming the cluster of negative charges responsible for binding cytochrome c change their accessibility to water-soluble protein modifier reagents; as a consequence the electrostatic field that controls the enzyme-substrate interaction is altered and cytochrome c appears to bind differently to oxidase; photolabeling experiments with the enzyme from bovine heart and other eukaryotic sources show that ATP cross-links specifically to the cytoplasmic subunits IV and VIII. Taken together, these data indicate that ATP can, at physiological concentration, bind to cytochrome c oxidase and induce an allosteric conformational change, thus affecting the interaction of the enzyme with cytochrome c. These findings raise the possibility that the oxidase activity may be influenced by the cell environment via cytoplasmic subunit-mediated interactions.