Coulometric and potentiometric evaluation of the redox components of cytochrome c oxidase in situ

A new coulometric-potentiometric titration cuvette is described which permits accurate measurements of oxidation-reduction components in membranous systems. This cuvette has been utilized to measure the properties of cytochrome c oxidase in intact membranes of pigeon breast muscle mitochondria. The reducing equivalents accepted and donated by the portion of the respiratory chain with half-reduction potentials greater than 200 mV are equal to those required for the known components (cytochrome a3 and the high-potential copper plus cytochrome a, 'visible copper', cytochrome c1, cytochrome c, and the Rieske iron-sulfur protein). Titrations in the presence of CO show that formation of the reduced cytochrome a3-CO complex requires two reducing equivalents per cytochrome a3 (coulometric titration). Potentiometric titrations indicate (Lindsay, J.G., Owen, C.S. and Wilson, D.F. (1975) Arch. Biochem. Biophys. 169, 492--505) that both cytochromes a3 and the high-potential copper must be reduced in order to form the CO complex (n = 2.0 with a CO concentration-dependent half-reduction potential, Em). By contrast, titrations in the presence of azide show that the Em value of the high-potential copper is unchanged by the presence of azide and thus azide binds with nearly equal affinity whether the copper is reduced or oxidized.     

The redox properties of the cytochromes of purified Complex III

Purified Complex III from beef heart contains two b cytochromes: a high-potential (E_{m 7.2} = +93 mV) cytochrome b-562 which can be enzymatically reduced, and a low-potential (E_{m 7.2} = −34 mV) cytochrome b-565 which is reduced only by dithionite. The two components each contribute approximately 50% to the total cytochrome b of Complex III. Cytochrome c₁ of Complex III titrates with a half-reduction potential of +232 mV.     

Mitochondrial respiratory chain of Tetrahymena pyriformis The properties of submitochondrial particles and the soluble b and c type pigments

Submitochondrial particles isolated from Tetrahymena pyriformis contain essentially the same redox carriers as those present in parental mitochondria: at pH 7.2 and 22 °C there are two b-type pigments with half-reduction potentials of −0.04 and −0.17 V, a c-type cytochrome with a half reduction potential of 0.215 V, and a two-component cytochrome a₂ with E_m7.2 of 0.245 and 0.345 V.

EPR spectra of the aerobic submitochondrial particles in the absence of substrate show the presence of low spin ferric hemes with g values at 3.4 and 3.0, a high spin ferric heme with g = 6, and a g = 2.0 signal characteristic of oxidized copper. In the reduced submitochondrial particles signals of various iron-sulfur centers are observed.

Cytochrome c₅₅₃ is lost from mitochondria during preparation of the submitochondrial particles. The partially purified cytochrome c₅₅₃ is a negatively charged protein at neutral pH with an E_m7.2 of 0.25 V which binds to the cytochrome c-depleted Tetrahymena mitochondria in the amount of 0.5 nmol/mg protein with a K_D of 0.8 · 10⁻⁶ M. Reduced cytochrome c₅₅₃ serves as an efficient substrate in the reaction with its own oxidase. The EPR spectrum of the partially purified cytochrome c₅₅₃ shows the presence of a low spin ferric heme with the dominant resonance signal at g = 3.28.

A pigment with an absorption maximum at 560 nm can be solubilized from the Tetrahymena cells with butanol. This pigments has a molecular weight of approx. 18 000, and E_m7.2 of −0.17 V and exhibits a high spin ferric heme signal at g = 6.     

Biochemical and biophysical studies on cytochrome c oxidase. XIX. An EPR study of the photodissociation of carboxycytochrome c oxidase in the presence of azide

1. The photodissociation reaction of the cytochrome c oxidase-CO compound in the presence of azide was studied by EPR at 15°K. Addition of CO in the dark to cytochrome c oxidase, partially reduced (2 electrons per 4 metal ions) in the presence of azide brings about a decrease in intensity of the azide-induced low-spin heme signal at g = 2.9, 2.2 and 1.67 and an increase in intensity of both the low-spin heme signal at g = 3 and the copper signal at g = 2. Subsequent illumination with white light at room temperature of this sample causes an enhancement of the azide-induced signal at g = 2.9, and a decrease in intensity of both signals at g = 3 and g = 2. It is shown that these changes in the EPR spectrum are reversible.

2. These results demonstrate that upon photodissociation, CO is replaced by azide wheras upon incubation in the dark CO expels azide from its binding site in cytochrome c oxidase.

3. Concomitantly with the binding of CO and dissociation of the azide molecule, and vice versa, electron redistributions occur as inferred from the changes in the intensity of the copper signal at g = 2.

4. The results are explained in a model of cytochrome c oxidase with either a common binding site (cytochrome a₃)^* for CO and azide or in a model with anti-cooperative interaction between two different sites of binding.

5. Similar types of experiments with cyanide instead of azide show that cyanide is more firmly bound to partially reduced cytochrome c oxidase than CO and azide. The affinity of ligands for partially reduced enzyme decreases in the sequence: cyanide, CO (dark), azide and CO (illuminated).     

Azide inhibition of mitochondrial electron transport II. Spectral changes induced by azide

Azide inhibition of coupled mitochondrial transport is accompanied by spectral changes which indicate that the cytochrome a₃ is oxidized and cytochrome a reduced. The cytochrome a absorption band is shifted to shorter wavelengths in the azideinhibited system. This shift in the absorption band can be reversed by conditions leading to reduction of cytochrome a₃ such as uncouplers and anaerobiosis, or terminal inhibitors such as sulfide, cyanide or CO.

Titrations of the azide-induced spectral changes indicate the binding of one azide molecule in the complex, and that the dissociation constant is experimentally indistinguishable from the uncompetitive inhibitor constants for inhibition of State 3 respiration. The azide inhibition is postulated to involve the formation of a reduced cytochrome a azide compound which is unstable in the presence of reduced cytochrome a₃.     

Properties of the cytochrome a-like material developed in the photosynthetic bacterium Rhodopseudomonas spheroides when grown aerobically

A photosynthetically-incompetent mutant Rhodopseudomonas spheroides that lacks bacteriochlorophyll was isolated. Spectroscopic evidence from CO difference spectra and cyanide difference spectra suggested that a cytochrome oxidase was present in this mutant that contained two components, corresponding to cytochromes a and a₃ of mitochondria. Potentiometric titration at 607 nm also showed the presence of two components with oxidation-reduction mid-point potentials of +375 mV and +200 mV. They were present in a ratio close to unity. No cytochrome of the the c-type corresponding to mitochondrial cytochrome c was detected, but a minor c component (near 10% of the total cytochrome c) with an oxidation-reduction mid-point potential of +120 mV was found

Growth of the mutant in medium with low aeration or lacking added copper diminished the concentration of the a-type cytochrome but not the concentrations of cytochromes of the b and c-type.     

Low-temperature spectral properties of the respiratory chain cytochromes of mitochondria from Crithidia fasciculata

Highly purified mitochondrial preparations from the trypanosomatid hemoflagellate, Crithidia fasciculata (A.T.C.C. No.11745), were examined by low-temperature difference spectroscopy. The cytochrome a+a₃ maximum of hypotonically-treated mitochondria reduced with succinate, was shifted from 605 nm at room temperature to 601 nm at 77 °K. The Soret maximum, found at 445 nm at 23 °C, was split at 77 °K into two approximately equally absorbing species with maxima at 438 and 444 nm. A prominent shoulder observed at 590 nm with hypotonically-treated mitochondria was not present in spectra of isotonic controls.

The cytochrome b maxima observed in the presence of succinate plus antimycin A were shifted from the 431 and 561 nm positions observed at 23 °C to 427 and 557 nm at 77 °K. Multiple b cytochromes were not apparent.

Unlike other soluble c-type cytochromes, the maximum of cytochrome c₅₅₅ was not shifted at 77 °K although it was split to give a 551 nm shoulder adjacent to the 555 nm maximum. This lack of a low-temperature blue shift was true for partially purified hemoprotein preparations as well as in situ in the mitochondrial membrane.

Using cytochrome c₅₅₅-depleted mitochondria, a cytochrome c₁ pigment was observed with a maximum at 420 nm and multiple maxima at 551, 556, and 560 nm. After extraction of non-covalently bound heme, the pyridine hemochromogen difference spectrum of cytochrome c₅₅₅-depleted preparations exhibited an maximum at 553 nm at room temperature.

The reduced rate of succinate oxidation by cytochrome c₅₅₅-depleted mitochondria and the ferricyanide requirement for the reoxidation of cytochrome c₁, even in the presence of antimycin, indicated that cytochrome c₅₅₅-mediated electron transfer between cytochromes c₁ and a+a₃ in a manner analagous to that of cytochrome c in mammalian mitochondria.     

Further studies on the NADH oxidase of the cytoplasmic membrane of Mycobacterium tuberculosis

The cytoplasmic membrane of the H₃₇Ra strain of Mycobacterium tuberculosis has been isolated free of cell wall.

These membrane preparations contain very small quantities of cytochromes c, b and cytochrome oxidase. The cytochrome c is not extracted by any method attempted. The cytochrome b is reducible only by dithionite and is believed not to be involved in the direct transfer of electrons during the oxidation of NADH by these preparations. The NADH oxidase activity of the membrane is inhibited by high concentrations of cyanide and also by 2-(n-heptyl)-4-hydroxyquinoline-N-oxide (HQNO). The cytochrome oxidase of the membrane contains both cytochromes a and a₃ and is present in low concentrations relative to cytochrome c. The cytochrome a₃ component was identified by characteristic complexes with both CO and cyanide and shows a γ-band absorption maximum at a slightly lower wavelength than the cytochrome oxidase of mammalian mitochondria (442 nm vs. 445 nm). The functional activity of the cytochrome oxidase is indicated by the inhibition of reoxidation of reduced cytochromes c and a in the presence of cyanide.     

Redox state of the partially reduced cytochrome aa3-cyanide complex

The low-spin ferric cyanide complex of beef heart cytochrome aa₃ can be partially reduced by stoichiometric additions of ferrous cytochrome c or by similar additions of N,N,N′,N′-tetramethyl-p-phenylene diamine. In both cases the initial ratio of cytochrome c oxidized: cytochrome a reduced or Wurster's Blue: cytochrome a reduced approximates the value 2. It is concluded that the binding of a single HCN prevents the reduction of both cytochrome a₃ and its associated EPR-invisible Cu atom.     

Physiological electron donors to the photochemical reaction center of Rhodobacter capsulatus

The nature and number of physiological electron donors to the photochemical reaction center of Rhodobacter capsulatus have been probed by deleting the genes for cytochromes c₁ and b of the cytochrome bc₁ complex, alone or in combination with deletion of the gene for cytochrome c₂. Deletion of cytochrome c₁ renders the organism incapable of photosynthetic growth, regardless of the presence or absence of cytochrome c₂, because in the absence of the bc₁ complex there is no cyclic electron transfer, nor any alternative source of electrons to rereduce the photochemically oxidized reaction center. While cytochrome c₂ is capable of reducing the reaction center, there appears no alternative route for its rereduction other than the bc₁ complex. The deletion of cytochromes c₁ and c₂ reveals previously unrecognized membrane-bound and soluble high potential c-type cytochromes, with E_m7 = + 312 mV and E_m6.5 = +316 mV, respectively. These cytochromes do not donate electrons to the reaction center, and their roles are unknown.     

Activation by reduction of the resting form of cytochrome c oxidase: Tests of different models and evidence for the involvement of CuB

(1) The reaction of the resting form of oxidised cytochrome c oxidase from ox heart with dithionite has been studied in the presence and absence of cyanide. In both cases, cytochrome a reduction in 0.1 M phosphate (pH 7) occurs at a rate of 8.2 · 10⁴ M⁻¹ · s⁻¹. In the absence of cyanide, ferrocytochrome a₃ appears at a rate (k_obs) of 0.016 s⁻¹. Ferricytochrome a₃ maintains its 418 nm Soret maximum until reduced. The rate of a₃ reduction is independent of dithionite concentration over a range 0.9 mM–131 mM. In the presence or cyanide, visible and EPR spectral changes indicate the formation of a ferric a₃/cyanide complex occurs at the same rate as a₃ reduction in the absence of cyanide. A g = 3.6 signal appears at the same time as the decay of a g = 6 signal. No EPR signals which could be attributed to copper in any significant amounts could be detected after dithionite addition, either in the presence or absence of cyanide. (2) Addition of dithionite to cytochrome oxidase at various times following induction of turnover with ascorbate/TMPD, results in a biphasic reduction of cytochrome a₃ with an increasing proportion of the fast phase of reduction occurring after longer turnover times. At the same time, the predominant steady state species of ferri-cytochrome a₃ shifts from high to low spin and the steady-state level of reduction of cytochrome a drops indicating a shift in population of the enzyme molecules to a species with fast turnover. In the final activated form, oxygen is not required for fast internal electron transfer to cytochrome a₃. In addition, oxygen does not induce further electron uptake in samples of resting cytochrome oxidase reduced under anaerobic conditions in the presence of cyanide. Both findings are contrary to predictions of certain O-loop types of mechanism for proton translocation. (3) A measurement of electron entry into the resting form of cytochrome oxidase in the presence of cyanide, using TMPD or cytochrome c under anaerobic conditions, shows that three electrons per oxidase enter below a redox potential of around +200 mV. An initial fast entry of two electrons is followed by a slow (k_obs ≈ 0.02 s) entry of a third electron. Above +200 mV, the number of electrons taken up in the initial fast phase drops as a redox center (presumably Cu_A) titrates with an apparent mid-point potential of +240 mV. The slow phase of reduction remains at the more positive redox values. (4) The results are interpreted in terms of an initial fast reduction of cytochrome a (and Cu_A at redox values more negative than +240 mV) followed by a slow reduction of Cu_B. Cu_B reduction is proposed to spin-uncouple cytochrome a₃ to form a cyanide sensitive center, and trigger a conformational change to an activated form of the enzyme with faster intramolecular electron transfer.     

Energy transduction in photosynthetic bacteria. XI. Further resolution of cytochromes of b type and the nature of the CO-sensitive oxidase present in the respiratory chain of Rhodopseudomonas capsulata

1. In membranes prepared from dark grown cells of Rhodopseudomonas capsulata, five cytochromes of b type (E′₀ at pH 7.0 +413±5, +270±5, +148±5, +56±5 and −32±5 mV) can be detected by redox titrations at different pH values. The midpoint potentials of only three of these cytochromes (b₁₄₈, b₅₆, and b₋₃₂) vary as a function of pH with a slope of 30 mV per pH unit.

2. In the presence of a Co/N₂ mixture, the apparent E′₀ of cytochrome b₂₇₀ shifts markedly towards higher potentials (+355 mV); a similar but less pronounced shift is apparent also for cytochrome b₁₅₀. The effect of CO on the midpoint potential of cytochrome b₂₇₀ is absent in the respiration deficient mutant M6 which possesses a specific lesion in the CO-sensitive segment of the branched respiratory chain present in the wild type strain.

3. Preparations of spheroplasts with lysozyme digestion lead to the release of a large amount of cytochrome c₂ and of virtually all cytochrome cc′. These preparations show a respiratory chain impaired in the electron pathway sensitive to low KCN concentration, in agreement with the proposed role of cytochrome c₂ in this branch; on the contrary, the activity of the CO-sensitive branch remains unaffected, indicating that neither cytochrome c₂ nor the CO-binding cytochrome cc′ are involved in this pathway.

4. Membranes prepared from spheroplasts still possess a CO-binding pigment characterized by maxima at 420.5, 543 and 574 nm and minima at 431, 560 nm in CO-difference spectra and with an band at 562.5 nm in reduced minus oxidized difference spectra. This membrane-bound cytochrome, which is coincident with cytochrome b₂₇₀, can be classified as a typical cytochrome “o” and considered the alternative CO-sensitive oxidase.     

Azide inhibition of mitochondrial electron transport I. The aerobic steady state of succinate oxidation

The azide inhibition of the succinate oxidase activity of rat-liver mitochondria is specific for active (State 3) respiration with no observable inhibition of resting (State 4) respiration. In the range of azide concentrations which inhibit State 3 to rates less than those of State 4, a negative control of respiration by ADP and inorganic phosphate is observed. The inhibition is specific for a site between cytochromes a and a₃, causing a crossover between these two cytochromes with cytochrome a becoming reduced and cytochrome a₃ remaining highly oxidized. Trapped steady-state difference spectra at liquid nitrogen temperatures show that the reduced cytochrome a in the azide-inhibited system has an band at 596 mμ, 6 m μ displaced from its usual position at 602 mμ.

The azide inhibition is released by uncouplers of oxidative phosphorylation such that the uncoupled respiration requires up to ten times as much azide as does coupled (State 3) respiration for comparable inhibition. The release of inhibition by uncouplers occurs with no change in the steady-state concentration of reduced cytochrome a₅₉₆ and the increased respiration is attributed to an increased rate of oxidation of the cytochrome a₅₉₆. This cytochrome is postulated to be either an intermediate in electron transport and energy conservation reactions or an azide compound of such an intermediate.     

Inhibition of respiration and destruction of cytochrome a3 by light in mitochondria and cytochrome oxidase from beef heart

Irradiation of beef-heart mitochondria and of cytochrome oxidase purified from beef-heart mitochondria with blue light inhibited electron transport from substrate (succinate for the mitochondria and reduced cytochrome c for the cytochrome oxidase) to O₂. The irradiation treatment also destroyed cytochrome a₃ as assayed by the absorption band for the reduced cyanide-cytochrome a₃ complex at 587 nm in the low-temperature absorption spectrum. Irradiation under anaerobic conditions was not inhibitory. Cytochrome a₃ was protected against photodestruction if cyanide was present during the irradiation.     

Cytochrome oxidase and its derivatives. VIII. Formation and properties of the ‘oxygenated’ from of cytochrome oxidase and its reconversion to ferrous and ferric oxidase

1. The ‘oxygenated’ compound of cytochrome c oxidase used in our experiments is more stable than the compound of previous reports. It is quantitatively reversible to ferrous oxidase.

2. It is best formed with an excess of O₂ after reduction with a minimum amount of dithionite. It can also be formed at low O₂ tension, but then contains some ferric oxidase.

3. Its formation from ferrocyanide-reduced oxidase remains incomplete and subsequent reduction by dithionite is also incomplete.

4. Cyanide does not inhibit its formation from ferrous oxidase. If only ferricytochrome a but no ferricytochrome a₃ is reduced in the presence of cyanide by dithionite, there is no reaction with O₂.

5. The anaerobic reduction of ‘oxygenated’ oxidase by dithionite is monophasic and fast. In contrast, that of ferric oxidase is biphasic, with an initial fast reduction of ferricytochrome a followed by a much slower reduction of ferricytochrome a₃. The rate of cytochrome a, but not that of cytochrome a₃ reduction depends on dithionite concentration.

6. In the presence of dissolved O₂, the ferric oxidase reduction comes to a temporary standstill when one-third of the absorbance increase at 444 mμ has been reached.

7. Ethyl hydrogen peroxide reacting with ferrous oxidase forms a compound similar to the ‘oxygenated’ compound.

8. Hydrogen donors known to react with peroxidase-H₂O₂ complexes, particularly pyrogallol, accelerate the transformation of ‘oxygenated’ to ferric oxidase, though not at a rate comparable to that of cytochrome c.

9. These results strengthen the evidence for cytochromes a and a₃ but indicate that this difference has disappeared in ‘oxygenated’ oxidase.     

Redistribution of electric charge accompanying photo-synthetic electron transport in Chromatium

The isoionic pH of Chromatium chromatophores is 5.2±0.1. At pH 7.7, the net charge on the chromatophore is approx. −1·10⁴. If a change in this charge accompanies the oxidation of an electron carrier, the midpoint redox potential (E_m) of that carrier should be a function of the solution ionic strength (I). of that carrier should be a function of the solution ionic strength (I).

The E_m values of P870 and cytochrome c-555 increase strongly with increasing I at low values of I. The E_m of cytochrome c-552 also increases with increasing I, though not so strongly. These effects probably cannot be attributed to an influence of I on the activity coefficient of a dissociable ion. We conclude that, when either P870 or cytochrome c-555 loses an electron, no specific ions (including protons) are bound or released in significant amounts, and the absolute value of the charge on the chromatophore decreases.

The E_m values of the primary and secondary electron acceptors, X and Y, do not depend on I. Because these E_m values have been shown previously to depend on pH, we conclude that the uptake of a proton keeps the charge on the chromatophore constant when either X or Y accepts an electron. This means that the primary and secondary electron transfer reactions in Chromatium result in a net decrease in the charge on the photosynthetic membrane. They do not result in the translocation of protons across the membrane.

The E_m of the soluble flavocytochrome c-552 from Chromatium depends only weakly on I, but depends strongly on the pH. The uptake of a proton appears to keep the net charge on this cytochrome constant upon reduction.     

Influence du pH sur la synthèse de l''oxidase (cytochrome a3) chez Bacillus coagulans, microorganisme thermophile, capable d'' “adaptation respiratoire”

In a new series of experiments on Bacillus coagulans (ATCC 11.369), it was demonstrated that this organism possesses a respiratory system with cytochromes b, c₁, c, (a+a₃) and also cytochrome o. A small decrease in the pH of the growth medium from 6.5 to 5.5 increases the respiratory activity by a factor of 4 and induces a variation of the absorption ratio [₆₀₃ (a+a₃)]/[₅₆₀ (b+c)] resulting in a preponderant increase in the ₆₀₃ absorption. The kinetic studies of the respiratory system synthesis during the phenomenon of “respiratory adaptation” have shown that lowering the pH of the adaption medium has the same effect. Spectral studies of membrane fractions (red dithionite) with or without carbon monoxide showed a preferential synthesis of oxidase a₃.     

The reaction of antimycin with a cytochrome b preparation active in reconstitution of the respiratory chain

1. Succinate-cytochrome c reductase activity was reconstituted by incubating a mixture of succinate dehydrogenase, cytochrome c₁, ubiquinone-10, phospholipid and a preparation of cytochrome b, made by the method of .

2. Preparations of cytochrome b active in reconstitution contained 5–28% native cytochrome b, as adjudged by reducibility with succinate in the reconstituted preparation and by lack of reaction with CO. Preparations of cytochrome b containing no native cytochrome b according to this criterion were inactive in reconstitution.

3. With a fixed amount of cytochrome b, the activity of the reconstituted preparation increased with increasing amounts of cytochrome c₁ until a ratio of about 2b (total): 1c₁ (allowing for the cytochrome c₁ present in the cytochrome b preparation) was reached.

4. The amount of antimycin necessary for maximal inhibition of the reconstituted enzyme is a function of the amount of the cytochrome b and is independent of the amount of cytochrome c₁. It is equal to about one half the amount of native cytochrome b.

5. Preparations of intact or reconstituted succinate-cytochrome c reductase or of cytochrome b completely quench the fluorescence of added antimycin, until an amount of antimycin equal to onehalf the amount of native cytochrome b present was added. Antimycin added in excess of this amount fluoresces with normal intensity. The quenching is only partial in the presence of Na₂S₂O₄. Denatured cytochrome b does not quench the fluorescence.

6. Since preparations of cytochrome b active in reconstitution contained cytochrome c₁ in an amount exceeding one half the amount of native cytochrome b present in the preparation, there is no evidence that native cytochrome b has been resolved from cytochrome c₁. The stimulatory action of cytochrome c₁ may be due to the restoration of a damaged membrane conformation.

7. Based on the assumption that the bc₁ segment of the respiratory chain contains 2b:1c₁:1 antimycin-binding sites, the specific quenching of antimycin fluorescence by binding to cytochrome b enables an accurate determination of the absorbance coefficients of cytochromes b and c₁. These are 25.6 and 20.1 mM⁻¹×cm⁻¹ for the wavelength pairs 563–577 nm and 553–539 nm, respectively, in the difference spectrum reduced minus oxidized.     

The respiratory chain of plant mitochondria. XV. Equilibration of cytochromes c549, b553, b557 and ubiquinone in mung bean mitochondria: Placement of cytochrome b557 and estimation of the midpoint potential of ubiquinone

1. 1. Cycles of oxidation followed by reduction at pH 7.2 have been induced in uncoupled anaerobic mung bean mitochondria treated with succinate and malonate by addition of oxygen-saturated medium. Under the conditions used, cytochromes b₅₅₇, b₅₅₃, c₅₄₉ (corresponding to c₁ in mammalian mitochondria) and ubiquinone are completely oxidized in the aerobic state, but become completely reduced in anaerobiosis.

2. 2. The time course of the transition from fully oxidized to fully reduced in anaerobiosis was measured for cytochromes c₅₄₉, b₅₅₇, and b₅₅₃. The intramitochondrial redox potential (IMP_h) was calculated as a function of time for each of the three cytochromes from the time course of the oxidized-to-reduced transition and the known midpoint potentials of the cytochromes at pH 7.2. The three curves so obtained are superimposable, showing that the three cytochromes are in redox equilibrium under these conditions during the oxidized-to-reduced transition.

3. 3. This result shows that the slow reduction of cytochrome b₅₅₇ under these conditions, heretofore considered anomalous, is merely a consequence of its more negative midpoint potential of +42 mV at pH 7.2, compared to +75 mV for cytochrome b₅₅₃ and +235 mV for cytochrome c₅₄₉. Cytochrome b₅₅₇ is placed on the low potential side of coupling site II and transfers electrons to cytochrome c₅₄₉ via the coupling site.

4. 4. The time course of the transition from fully oxidized to fully reduced was also measured for ubiquinone. Using the change in intramitochondrial potential IMP_h with time obtained from the three cytochromes, the change in redox state of ubiquinone with IMP_h was calculated. When replotted as IMP_h versus the logarithm of the ratio (fraction oxidized)/(fraction reduced), two redox components with n = 2 were found. The major component is ubiquinone with a midpoint potential E_m7.2 = + 70 mV. The minor component has a midpoint potential E_m7.2 = − 12 mV; its nature is unknown.

Biochemical and biophysical studies on cytochrome c oxidase XIV. The reaction with cytochrome c as studied by pulse radiolysis

1. The reduction of cytochrome c oxidase by hydrated electrons was studied in the absence and presence of cytochrome c.

2. Hydrated electrons do not readily reduce the heme of cytochrome c oxidase. This observation supports our previous conclusion that heme a is not directly exposed to the solvent.

3. In a mixture of cytochrome c and cytochrome c oxidase, cytochrome c is first reduced by hydrated electrons (k = 4 · 10¹⁰ M⁻¹ · s⁻¹ at 22 °C and pH 7.2) after which it transfers electrons to cytochrome c oxidase with a rate constant of 6 · 10⁷ M⁻¹ · s⁻¹ at 22 °C and pH 7.2.

4. It was found that two equivalents of cytochrome c are oxidized initially per equivalent of heme a reduced, showing that one electron is accepted by a second electron acceptor, probably one of the copper atoms of cytochrome c oxidase.

5. After the initial reduction, redistribution of electrons takes place until an equilibrium is reached similar to that found in redox experiments of Tiesjema, R. H., Muijsers, A. O. and Van Gelder, B. F. (1973) Biochim. Biophys. Acta 305, 19–28.