The use of principal component analysis to resolve the spectra and kinetics of cytochrome c oxidase reduction by 5,10-dihydro-5-methyl phenazine.

The method of principal component analysis (PCA) was applied to the absorption-wavelength-time surfaces generated by rapid scanning stopped-flow spectrophotometry (RSSFS). The method was used to resolve the absorption surfaces generated during the reduction of cytochrome c oxidase by 5,10-dihydro-5-methyl phenazine (MPH) into the individual spectral shapes and time courses of the component chromophores. Two forms of resting cytochrome oxidase were used in these analyses: one that has its maximum absorption in the Soret region at 418 nm (418-nm species) and the other has its absorption maximum at 424 nm (424-nm species). A weighting scheme suitable for RSSFS data was developed. The optical absorption spectra obtained by W.H. Vanneste (1966, Biochemistry, 5:838-848) for the oxidase components were found to fit adequately as components of the experimental surfaces. Among these spectra were the oxidized forms of cytochromes a and a3 in the wavelength region 330-520 nm for the 418-nm species. Vanneste's spectral shape for the oxidized cytochrome a3 did not fit as a component in the spectrum of the 424-nm species. After accounting for the spectral shape of all components present, PCA provided a straightforward method for determining the separate time courses of each chromophore. We have found for both forms used that cytochrome a is reduced by MPH in the initial stages of the reaction, while cytochrome a3 is reduced in subsequent, slow phases. An important aspect of PCA is that it provided confirmation of the spectra of the various oxidase components without requiring the use of inhibitors or the use of simplifying mechanistic assumptions. The resolution of time profiles of strongly overlapping chromophores is also demonstrated.     

Reactions of mercaptans with cytochrome c oxidase and cytochrome c

1. The steady-state oxidation of ferrocytochrome c by dioxygen catalyzed by cytochrome c oxidase, is inhibited non-competitively towards cytochrome c by methanethiol, ethanethiol, 1-propanethiol and 1-butanethiol with Ki values of 4.5, 91, 200 and 330 microM, respectively. 2. The inhibition constant Ki of ethanethiol is found to be constant between pH 5 and 8, which suggests that only the neutral form of the thiol inhibits the enzyme. 3. The absorption spectrum of oxidized cytochrome c oxidase in the Soret region shows rapid absorbance changes upon addition of ethanethiol to the enzyme. This process is followed by a very slow reduction of the enzyme. The fast reaction, which represents a binding reaction of ethanethiol to cytochrome c oxidase, has a k1 of 33 M-1 . s-1 and a dissociation constant Kd of 3.9 mM. 4. Ethanethiol induces fast spectral changes in the absorption spectrum of cytochrome c, which are followed by a very slow reduction of the heme. The rate constant for the fast ethanethiol reaction representing a bimolecular binding step is 50 M-1 . s-1 and the dissociation constant is about 2 mM. Addition of up to 25 mM ethanethiol to ferrocytochrome c does not cause spectral changes. 5. EPR (electron paramagnetic resonance) spectra of cytochrome c oxidase, incubated with methanethiol or ethanethiol in the presence of cytochrome c and ascorbate, show the formation of low-spin cytochrome alpha 3-mercaptide compounds with g values of 2.39, 2.23, 1.93 and of 2.43, 2.24, 1.91, respectively.     

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 8.2.10(4) M-1.s-1. In the absence of cyanide, ferrocytochrome a3 appears at a rate (kobs) 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 (kobs approximately 0.02 s) entry of a third electron.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reaction of hydrogen peroxide with the rapid form of resting cytochrome oxidase.

The hydrogen peroxide binding reaction has been examined with alkaline-purified resting enzyme in order to avoid mixtures of low pH induced fast and slow conformers. At pH 8.8-9.0 (20 degrees C), the reactivity of resting enzyme was similar to the peroxide-free, pulsed conformer that has been characterized by other investigators. The reaction showed single-phase reactivity at 435 and 655 nm and required a minimum 8:1 molar excess of peroxide (over cytochrome a3) for quantitative reaction. At 16:1, the Soret band was stable for 1.0-1.5 h, but above 80:1, the band began showing generalized attenuation within 1-2 min. The peroxide binding reaction was also associated with an increase in absorbance at 606 nm which correlated with the rate of change at 435 and 655 nm. The observed rate constants at each of these wavelengths showed similar linear dependence on peroxide concentration, giving an average bimolecular rate constant of 391 M-1.s-1 and a Kd of 5.1 microM. The rise phase at 606 nm was observed to saturate at an 8:1 molar excess of peroxide but showed a slow, concentration-dependent first-order decay that gave a bimolecular rate constant and Kd of 38 M-1.s-1 and 20 microM, respectively. The decay was not associated with a change in the Soret absorption or charge-transfer regions, suggesting a type of spectral decoupling. An isosbestic point at 588 nm was consistent with the 606- to 580-nm conversion proposed by other investigators, although direct observation of a new band at 580 nm was difficult.(ABSTRACT TRUNCATED AT 250 WORDS)     

An analysis of the reaction kinetics of the hexahaem nitrite reductase of the anaerobic rumen bacterium Wolinella succinogenes.

The reduction kinetics of both the resting and redox-cycled forms of the nitrite reductase from the anaerobic rumen bacterium Wolinella succinogenes were studied by stopped-flow reaction techniques. Single-turnover reduction of the enzyme by dithionite occurs in two kinetic phases for both forms of the enzyme. When the resting form of the enzyme is subjected to a single-turnover reduction by dithionite, the slower of the two kinetic phases exhibits a hyperbolic dependence of the rate constant on the square root of the reductant concentration, the limiting value of which (approximately 4 s-1) is assigned to a slow internal electron-transfer process. In contrast, when the redox-cycled form of the enzyme is reduced by dithionite in a single-turnover experiment, both kinetic phases exhibit linear dependences of the rate on the square root of dithionite concentration, with associated rate constants of 150 M-1/2.s-1 and 6 M-1/2.s-1. Computer simulations of both the reduction processes shows that no unique set of rate constants can account for the kinetics of both forms, although the kinetics of the redox-cycled species is consistent with a much enhanced rate of internal electron transfer. Under turnover conditions the time course for reduction of the enzyme, in the presence of millimolar levels of nitrite and 100 mM-dithionite, is extremely complex. A working model for the mechanism of the turnover activity of the enzyme is proposed which very closely describes the reaction kinetics over a wide range of substrate concentrations, as shown by computer simulation. The similarity in the action of the nitrite reductase enzyme and mammalian cytochrome c oxidase is commented upon.     

Control of electron transfer by the electrochemical potential gradient in cytochrome-c oxidase reconstituted into phospholipid vesicles

The kinetics of electron transfer between cytochrome-c oxidase and ruthenium hexamine has been characterized using the native enzyme or its cyanide complex either solubilized by detergent (soluble cytochrome oxidase) or reconstituted into artificial phospholipid vesicles (cytochrome oxidase-containing vesicles). Ru(NH3)2+6 (Ru(II] reduces oxidized cytochrome a, following (by-and-large) bimolecular kinetics; the second order rate constant using the cyanide complex of the enzyme is 1.5 x 10(6) M-1 s-1, for the enzyme in detergent, and slightly higher for COV. In the case of COV the kinetics are not affected by the addition of ionophores. Upon mixing fully reduced cytochrome oxidase with oxygen (in the presence of excess reductants), the oxidation leading to the pulsed enzyme is followed by a steady state phase and (eventually) by complete re-reduction. When the concentrations of dioxygen and oxidase are sufficiently low (micromolar range), the time course of oxidation can be resolved by stopped flow at room temperature, yielding an apparent bimolecular rate constant of 5 x 10(7) M-1 s-1. After exhaustion of oxygen and end of steady state, re-reduction of the pulsed enzyme by the excess Ru(II) is observed; the concentration dependence shows that the rate of re-reduction is limited at 3 s-1 in detergent; this limiting value is assigned to the intramolecular electron transfer process from cytochrome a-Cua to the binuclear center. Using the reconstituted enzyme, the internal electron transfer step is sensitive to ionophores, increasing from 2-3 to 7-8 s-1 upon addition of valinomycin and carbonyl cyanide m-chlorophenylhydrazone. This finding indicates for the first time an effect of the electrochemical potential across the membrane on the internal electron transfer rate; the results are compared with expectations based on the hypothesis formulated by Brunori et al. (Brunori, M., Sarti, P., Colosimo, A., Antonini, G., Malatesta, F., Jones, M.G., and Wilson, M.T. (1985) EMBO J. 4, 2365-2368), and their bioenergetic relevance is discussed with reference to the proton pumping activity of the enzyme.     

Effect of heat treatment on oxidase activity and proton-pumping capability of proteoliposome-incorporated beef heart cytochrome aa3

By incubating beef heart cytochrome c oxidase at 43-45 degrees C, selective inactivation of the H+-pumping function is possible without affecting cytochrome c oxidase activity; proteoliposomes reconstituted with heated enzyme (43.5 degrees C for 60 min at pH 7.0) showed an apparent H+/e- ratio of only 0.3 and a turnover with cytochrome c plus ferrocyanide as substrate of 20 s-1, while those with the intact enzyme showed an apparent H+/e- ratio somewhat greater than 1.0 and a turnover of 19 s-1. This decrease in the H+/e- ratio could not be attributed to a stimulation of H+ permeability upon heating, since the respiratory control ratio and the magnitude of membrane potential formation remained almost the same in the two cases. A pH-dependent Em (midpoint redox potential) change of cytochrome a in the presence of cyanide was still observed after the heat treatment. Heating induced a small spectral shift in the Soret region of the oxidized (resting) enzyme; the peak of the heated enzyme was at 421 nm, while that of the intact enzyme was at 419 nm. The spectral shift obtained by pulsing the enzyme with oxygen under turnover conditions is also altered.     

Kinetic studies on oxidized and partially reduced cytochrome c oxidase.

1. Kinetic studies have been performed with beef-heart cytochrome c oxidase, with the enzyme either in its oxidized, resting state or pretreated anaerobically with different amounts of reduced cytochrome c. The techniques used for the study have been stopped-flow spectrophotometry and electron paramagnetic resonance (EPR) spectroscopy. 2. The results show that the one-electron equivalent-reduced enzyme rapidly oxidizes one further equivalent of aerobically or anaerobically added ferrocytochrome c, with a rate constant of 5 . 10(6) M-1 . s-1. 3. When an excess of ferrocytochrome c in the presence of oxygen is added to the one-electron-reduced enzyme, the same turnover rate is obtained as in experiments with the resting enzyme. 4. The one-electron equivalent-enzyme reacts with CO with a rate constant of 4 . 10(4) M-1 . s-1 to yield approx. 35% of the CO compound as compared with the reaction between the fully reduced enzyme and CO. 5. It is shown that on reduction the enzyme is converted into an active form, but it is concluded that the enzyme does not have to be fully reduced before it is catalytically active.     

Magnetic state of the alpha 3 center of cytochrome c oxidase and some of its derivatives.

The temperature dependence of the magnetic susceptibility was used to investigate the nature of the coupling between cytochrome alpha 3 and CuB in resting and oxidized cyanide- and formate-bound cytochrome oxidase. Resting and formate-bound enzymes were found to have strong antiferromagnetic coupling with an S = 5/2 cytochrome alpha 3, results that were independent of the dispersing detergent and the enzyme isolation method. The cyanide-bound enzyme was heterogeneous, with a minor fraction showing intermediate strength antiferromagnetic coupling. The magnitude of this coupling was independent of the enzyme isolation method and depended moderately on the identity of the dispersing detergent. The major fraction of the cyanide-bound enzyme had a lowest energy state of Ms = 0. The coupling constant for this fraction did not depend on the isolation technique or on the identity of the dispersing detergent. The use of glucose-glucose oxidase to deoxygenate samples influenced the susceptibility behavior of some preparations of both the resting and formate-bound enzymes, with results indicating an S = 3/2 cytochrome alpha 3 in the resting enzyme samples. Retention of a 417-nm Soret band for formate-bound enzyme concomitant with peroxide-induced changes in susceptibility behavior indicates different sites of enzyme interactions for the formate ion and hydrogen peroxide.     

Effect of changing the detergent bound to bovine cytochrome c oxidase upon its individual electron-transfer steps

The influence of the detergent environment upon individual electron-transfer rates of cytochrome c oxidase was investigated by stopped-flow spectrophotometry. The effects of three detergents were studied: lauryl maltoside, which supports a high turnover number (TN = 350 s-1), n-dodecyl octaethylene glycol monoether (C12E8), which supports an intermediate TN (150 s-1), and Triton X-100 in which oxidase is nearly inactive (TN = 2-3 s-1). Under limited turnover conditions (cytochrome c:cytochrome c oxidase ratio = 1:1 to 8:1), the rate of oxidation of cytochrome c was measured and compared with the fast reduction of cytochrome a and its relatively slow reoxidation. Two reducing equivalents of cytochrome c were rapidly oxidized in a burst phase; the remaining two to six equivalents were oxidized more slowly, concurrent with the reoxidation of cytochrome a; i.e., the percent reduced cytochrome a reflects the percent reduced cytochrome c. With the resting enzyme, the bimolecular reaction between reduced cytochrome c and cytochrome a was rapid, was insensitive to the detergent environment, and was not the rate-limiting step in the presence of any detergent. The rate of internal electron transfer from cytochrome a to cytochrome a3 in the resting enzyme was slow and only slightly affected by the detergent environment: 1.0-1.1 s-1 in Triton X-100, 5-7 s-1 in C12E8, and 5-12 s-1 in lauryl maltoside. With the pulsed enzyme, the intramolecular electron transfer between cytochrome a and cytochrome a3 increased 4-5-fold in the lauryl maltoside enzyme but did not increase in the Triton X-100 enzyme (intermediate values were obtained with the C12E8 enzyme). We conclude that cytochrome c oxidase acquires the pulsed conformation only in those detergents that support high TN's, e.g., lauryl maltoside and C12E8, but it is locked in the resting conformation in those detergents which result in low TN's, e.g., Triton X-100.     

Reduction of oxygen-pulsed cytochrome c oxidase by cytochrome c and other electron donors

1. Stopped-flow experiments were performed in which solutions containing dithionite were mixed with air-saturated buffer. Cytochrome c oxidase present in the dithionite-containing syringe is fully oxidized within the mixing time and the oxygen-pulsed form of the oxidase is produced. 2. The reduction of this form by dithionite, by dithionite plus cytochrome c and by dithionite plus methyl viologen or benzyl viologen was followed and compared with the corresponding reduction reactions of the "resting" oxidized enzyme. Reduction by dithionite is relatively slow, but the rate of reduction is greatly increased by addition of cytochrome c or the viologens, which are even more effective than cytochrome c on a molar basis. 3. Profound differences between the transient kinetics of the reduction of the two oxidized oxidase derivatives were observed. The results are consistent with a direct reduction of cytochrome a followed by an intramolecular electron transfer to cytochrome a3 (k1obs = 7.5 s-1 for the oxygen-pulsed oxidase). 4. The spectrum of the oxygen-pulsed oxidase formed within 5 ms of the mixing closely resembles that of the "oxygenated" compound, but there were small differences between the two spectra.     

Reaction of dioxygen with cytochrome c oxidase reduced to different degrees: indications of a transient dioxygen complex with copper-B.

The reactions of the fully reduced, three-electron-reduced, and mixed-valence cytochrome oxidase with molecular oxygen have been followed in flow-flash experiments, starting from the CO complexes, at 445 and 830 nm at pH 7.4 and 25 degrees C. With the fully reduced and the three-electron-reduced enzyme, four kinetic phases with rate constants in the range from 1 x 10(5) to 10(3) s-1 can be observed. The initial fast phase is associated with an absorbance increase at 830 nm. This is followed by an absorbance decrease (2.8 x 10(4) s-1), the amplitude of which increases with the degree of reduction of the oxidase. The third phase (6 x 10(3) s-1) displays the largest absorbance change at both wavelengths in the fully reduced enzyme and is not seen in the mixed-valence oxidase at 830 nm; a change with opposite sign but with a similar rate constant is found at 445 nm in this enzyme form. The slowest phase (10(3) s-1) is also largest in the fully reduced oxidase and not seen in the mixed-valence enzyme. It is suggested that O2 initially binds to reduced CuB and is then transferred to cytochrome a3 before electron transfer from cytochrome a/CuA takes place. The fast oxidation of cytochrome a seen with the fully reduced enzyme is suggested not to occur during natural turnover. A reaction cycle for the complete turnover of the enzyme is presented. In this cycle, the oxidase oscillates between electron input and output states of the proton pump, characterized by cytochrome a having a high and a low reduction potential, respectively.     

A new ruthenium complex to study single-electron reduction of the pulsed O(H) state of detergent-solubilized cytochrome oxidase

The first step in the catalytic cycle of cytochrome oxidase, the one-electron reduction of the fully oxidized enzyme, was investigated using a new photoactive binuclear ruthenium complex, [Ru(bipyrazine)2]2(quaterpyridine), (Ru2Z). The aim of the work was to examine differences in the redox kinetics resulting from pulsing the oxidase (i.e., fully reducing the enzyme followed by reoxidation) just prior to photoreduction. Recent reports indicate transient changes in the redox behavior of the metal centers upon pulsing. The new photoreductant has a large quantum yield, allowing the kinetics data to be acquired in a single flash. The net charge of +4 on Ru2Z allows it to bind electrostatically near CuA in subunit II of cytochrome oxidase. The photoexcited state Ru(II*) of Ru2Z is reduced to Ru(I) by the sacrificial electron donor aniline, and Ru(I) then reduces CuA with yields up to 60%. A stopped-flow-flash technique was used to form the pulsed state of cytochrome oxidase (the "OH" state) from several sources (bovine heart mitochondria, Rhodobacter sphaeroides, and Paracoccus denitrificans). Upon mixing the fully reduced anaerobic enzyme with oxygenated buffer containing Ru2Z, the oxidized OH state was formed within 5 ms. Ru2Z was then excited with a laser flash to inject one electron into CuA. Electron transfer from CuA --> heme a --> heme a3/CuB was monitored by optical spectroscopy, and the results were compared with the enzyme that had not been pulsed to the OH state. Pulsing had a significant effect in the case of the bovine oxidase, but this was not observed with the bacterial oxidases. Electron transfer from CuA to heme a occurred with a rate constant of 20,000 s-1 with the bovine cytochrome oxidase, regardless of whether the enzyme had been pulsed. However, electron transfer from heme a to the heme a3/CuB center in the pulsed form was 63% complete and occurred with biphasic kinetics with rate constants of 750 s-1 and 110 s-1 and relative amplitudes of 25% and 75%. In contrast, one-electron injection into the nonpulsed O form of the bovine oxidase was only 30% complete and occurred with monophasic kinetics with a rate constant of 90 s-1. This is the first indication of a difference between the fast form of the bovine oxidase and the pulsed OH form. No reduction of heme a3 is observed, indicating that CuB is the initial electron acceptor in the one-electron reduced pulsed bovine oxidase.     

Conformational change accompanies redox reactions of the tetraheme cytochrome c-554 of Nitrosomonas europaea

Cytochrome c-554 of the ammonia-oxidizing chemolithoautotropic bacteria is thought to mediate electron transfer from hydroxylamine oxidoreductase to a terminal oxidase and/or to ammonia monooxygenase. The cytochrome has four c hemes which interact magnetically and have the same redox potential. We report that the kinetics of reduction of ferric cytochrome c-554 by dithionite or the oxidation of ferrous cytochrome c-554 by O2 or H2O2 are complex and multiphasic. Transient rapid-scan difference spectra indicate discrete maxima at approximately 418 nm, 425 nm and 432 nm. Absorbance changes at all three difference maxima appear to occur in all kinetic phases, although not in equal amounts for each wavelength. Reduction by 20 mM dithionite was biphasic. At pH 7.5 the first phase, which involved approximately 50% of the total absorbance change, had a rate constant (20 degrees C) of 140 s-1 and energy of activation of 20 kJ X mol-1. The slow phase had a rate constant 0.43 s-1 and a relatively high energy of activation, 87 kJ X mol-1, suggesting that a change in protein configuration accompanied the reaction. As the pH of the solution increased, the rate constant for both phases decreased and the fraction of absorbance change in the rapid phase increased. Oxidation of ferrous cytochrome c-554 by O2 involved a discrete rapid phase with a rate constant of 14 s-1, accounting for 6% of the absorbance. The remainder of the reaction was multiphasic with rate constants in the range 0.1-0.01 s-1. With H2O2 as the oxidant, the rapid phase involved 39% of the change in absorbance with a rate constant of 19 s-1. The remainder of the reoxidation was multiphasic with rate constants ranging over 0.4-0.01 s-1.     

Spectral and kinetic abnormality during the reduction of cytochrome c3 catalyzed by hydrogenase with hydrogen

Reduction process of cytochrome c3 by hydrogenase (EC 1.12.2.1) under H2 was analyzed by means of spectrophotometry. When cytochrome c3 is in equilibrium with H2 under reduced pressure, spectral abnormality in the Soret region appeared most significantly in 1/4 reduction state, less significantly at 1/2 reduction state, and disappeared at 3/4 reduction state. The spectral changes during the enzymic reduction of cytochrome c3 in H2-saturated solution traced at several wavelengths also showed spectral abnormality in the Soret region at the early stage of reaction. The first-order rate constants for the successive reduction steps from all-ferric to all-ferrous form of cytochrome c3 was estimated to be k1 = 0.061 s-1, k2 = 0.063 s-1, k3 = 0.039 s-1 and k4 = 0.014 s-1 (cytochrome c3: 2 microM; hydrogenase: 2 nM, and at 20 degrees C, pH 7.0). Strong interaction is suggested between hemes 3 and 4 (for the refined structure and heme-numbering, see Higuchi, Y., Kusunoki, M., Matsuura, Y., Yasuoka, N. and Kakudo, M. (1984) J. Mol. Biol. 172, 109-139). The first electron from hydrogenase is supposed to be transferred to these hemes and delocalized between them, and the second electron, among hemes 3, 4 and 1. The characteristic behavior in the enzymic reduction of cytochrome c3 is different from that in non-enzymic reduction.     

Kinetic investigations of the reactions of cytochrome c oxidase with hydrogen peroxide

The reaction of H2O2 with reduced cytochrome c oxidase was investigated with rapid-scan/stopped-flow techniques. The results show that the oxidation rate of cytochrome a3 was dependent upon the peroxide concentration (k = 2 X 10(4) M-1 X s-1). Cytochrome a and CuA were oxidised with a maximal rate of approx. 20 s-1, indicating that the rate of internal electron transfer was much slower with H2O2 as the electron acceptor than with O2 (k greater than or equal to 700 s-1). Although other explanations are possible, this result strongly suggests that in the catalytic cycle with oxygen as a substrate the internal electron-transfer rate is enhanced by the formation of a peroxo-intermediate at the cytochrome a3-CuB site. It is shown that H2O2 took up two electrons per molecule. The reaction of H2O2 with oxidised cytochrome c oxidase was also studied. It is shown that pulsed oxidase readily reacted with H2O2 (k approximately 700 M-1 X s-1). Peroxide binding is followed by an H2O2-independent conformational change (k = 0.9 s-1). Resting oxidase partially bound H2O2 with a rate similar to that of pulsed oxidase; after H2O2 binding the resting enzyme was converted into the pulsed conformation in a peroxide-independent step (k = 0.2 s-1). Within 5 min, 55% of the resting enzyme reacted in a slower process. We conclude from the results that oxygenated cytochrome c oxidase probably is an enzyme-peroxide complex.     

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.     

Cyanide binding to bovine heart cytochrome c oxidase depleted of subunit III by treatment with lauryl maltoside

Subunit III was removed from beef heart cytochrome oxidase by incubation of the isolated enzyme at 25 degrees C for 24 h in lauryl maltoside buffer at a detergent to protein ratio of 10:1 (w:w). During the course of the incubation, the reaction of the enzyme with cyanide was followed by spectrophotometry in the Soret region. The starting material binds cyanide in a multiexponential process with 70% of the reaction occurring during the slow phase of the reaction at an observed rate of 3.85 X 10(-5) S-1 with 1 mM KCN. More of the enzyme binds cyanide during the fast phase of the reaction at an observed rate of 3.8 X 10(-3) S-1 as subunit III is removed by lauryl maltoside. After 24 h of incubation in lauryl maltoside, the enzyme reacts with cyanide completely in a rapid, single exponential process. When the protein from such an incubation is recovered by cytochrome c affinity chromatography and analyzed for its subunit content, subunit III is absent. The position of the Soret maximum of the oxidized enzyme shifts from its maximum at 418 nm in the starting material to 422 nm in the subunit III-depleted enzyme. The subunit III-depleted enzyme binds cyanide completely in a simple bimolecular reaction with a rate constant of 3.8 M-1 S-1. We discuss this result in terms of the possible structural and functional roles for subunit III in the cytochrome oxidase complex.     

The interactions between cytochrome c and cytochrome oxidase that determine the conformation of the oxidized oxidase.

1. Cytochrome c2+ increases the rate at which cytochrome oxidase (EC 1.9.3.1) gamma max428nm) converts to its conformational isomer (gamma max 418-423 nm) but cytochrome c3+ has little effect on the conversion rate. 2. Interactions between reduced cytochrome oxidase and cytochrome c were studied in the absence of electron flow using anaerobic Sephadex columns. 3. Oxidase that is reduced by cytochrome c2+ or other reductant forms the 418-to 423-nm isomer if its last contact, before oxidation, is with cytochrome c3+. If the reduced oxidase contacts cytochrome c2+, before oxidation, the 428-nm oxidase forms.     

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.