The effect of pH on the oxygen kinetics of cytochrome c oxidase proteoliposomes

The effect of pH on the oxygen kinetics of cytochrome c oxidase incorporated into phospholipid vesicles is studied. The pH profiles of the oxygen kinetics of energized and deenergized oxidase vesicles are similar. An effect of pH on the slope of the reciprocal plot of rate against oxygen concentration is observed, and this may indicate that protons are involved in the rate limiting step of the reaction between oxygen and reduced oxidase. In contrast to the pH dependence of the oxygen kinetics, the binding of CO to the oxidase is not pH dependent.     

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)     

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, E_m). By contrast, titrations in the presence of azide show that the E_m 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.     

Separation, stability and kinetics of monomeric and dimeric bovine heart cytochrome c oxidase

The stability of monomeric and dimeric bovine heart cytochrome c oxidase in laurylmaltoside-containing buffers of high ionic strength allowed separation of the two forms by gel-filtration high-performance liquid chromatography (HPLC). A solution of the dimeric oxidase could be diluted without monomerisation. Both monomeric and dimeric cytochrome c oxidase showed biphasic steady-state kinetics when assayed spectrophotometrically at low ionic strength. Thus, the biphasic kinetics did not result from negative cooperativity between the two adjacent cytochrome c binding sites of the monomers constituting the dimeric oxidase. On polyacrylamide gels in the presence of sodium dodecyl sulphate (SDS) a fraction of subunit III of the dimeric enzyme migrated as a dimer, a phenomenon not seen with the monomeric enzyme. This might suggest that in the dimeric oxidase subunit III lies on the contact surface between the protomers. If so, the presumably hydrophobic interaction between the two subunits III resisted dissociation by SDS to some extent. Addition of sufficient ascorbate and cytochrome c to the monomeric oxidase to allow a few turnovers induced slow dimerisation (on a time-scale of hours). This probably indicates that one of the transient forms arising upon reoxidation of the reduced enzyme is more easily converted to the dimeric state than the resting enzyme. Gel-filtration HPLC proved to be a useful step in small-scale purification of cytochrome c oxidase. In the presence of laurylmaltoside the monomeric oxidase eluted after the usual trace contaminants, the dimeric Complex III and the much larger Complex I. The procedure is fast and non-denaturing, although limited by the capacity of available columns.     

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 K_i values of 4.5, 91, 200 and 330 μM, respectively.2. The inhibition constant K_i 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 dissociation constant K_d 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 a₃-mercaptide compounds with g values of 2.39, 2.23, 1.93 and of 2.43, 2.24, 1.91, respectively.     

The use of specific lysine modifications to locate the reaction site of cytochrome c with sulfite oxidase

The reduction of cytochrome c by beef liver sulfite oxidase was found to be strongly inhibited by high ionic strength, indicating the importance of electrostatic interactions to the reaction. The reaction rates of sulfite oxidase with singly trifluoroacetylated or trifluoromethylphenylcarbamylated cytochrome c derivatives were studied to determine the role of individual lysines in the reaction. The reaction rate was decreased by modification of the lysines immediately surrounding the heme crevice, the decreases following the order: Lys 13 > Lys 25 Lys 79 ≈ Lys 87 > Lys 8 ≈ Lys 27 ≈ Lys 72. Modification of lysines 22, 55, 88, 99, and 100 had no effect on the reaction rate. These results indicate that the interaction site on cytochrome c for sulfite oxidase is at the heme crevice region, and overlaps considerable with that for cytochrome oxidase.     

The intrinsic stability of the second intermediate following the dioxygen-bound form in the O2 reduction by cytochrome c oxidase

Aeration of a two-electron reduced cytochrome c oxidase provides a species with two Raman bands at 804 and 356 cm⁻¹, identifying it as the second intermediate following the O₂-bound species in the enzymatic O₂ reduction process. It degrades directly to the fully oxidized form with a half-life time of 70 min at pH 8.0. The stability suggests an effective insulation for the active site in an extremely high oxidation state (Fe⁴⁺ with one oxidative equivalent nearby) against spontaneous electron leaks, which would dissipate proton motive force. The formation and degradation of the second intermediate are pH-dependent.     

A 1H-NMR longitudinal relaxation study of the interaction between cytochrome c and cytochrome c oxidase

The interaction between the oxidized forms of cytochrome c and cytochrome c oxidase (EC 1.9.3.1) has been investigated by ¹H-NMR longitudinal relaxation measurements. It is found that relaxation of methyl groups on the heme ring of cytochrome c markedly deviates from a simple exponential behavior in the presence of small amounts of cytochrome oxidase. A comparison with the relaxation behavior of cytochrome c modified by 4-carboxy-3,5-dinitrophenyl at Lys-13 shows that the oxidase induces a conformation in native cytochrome c that is closely related to that of the derivative. It is suggested that this change in conformation consists of a rupture of the salt bridge between Lys-13 and Glu-90 and a concomitant perturbation of the methionine ligand.     

Lipid specificity for membrane mediated partial unfolding of cytochrome c

In this study we investigated the lipid specificity for destabilization of the native structure of horse heart cytochrome c by model membranes. From (i) the enhanced release of deuterium from deuterium-labelled cytochrome c and (ii) the increased proteolytic digestion of the protein in the presence of anionic lipids, it was concluded that these lipids are able to destabilize the native structure of cytochrome c. Changes in the absorbance at 695 nm indicated that the destabilization was accompanied by a diminished ligation of Met-80 to the heme. Beef heart cardiolipin was found to be more effective than DOPS, DOPG or DOPA, while no protein destabilization was observed in the presence of the zwitterionic lipid DOPC or, surprisingly, in the presence of E. coli cardiolipin. Experimnts with mitoplasts showed that the protein can also be destabilized in its native structure by a biological membrane.     

The role of a cytochrome c-552-cytochrome c complex in the oxidation of sulfide in Chromatium vinosum

The sulfide:cytochrome c oxidoreductase activity of the flavocytochrome c-522 from the purple sulfur bacterium Chromatium vinosum has been investigated. The oxidized sulfur product of the sulfide:cytochrome c reductase activity has been shown to be elemental sulfur. Cytochrome c-552 has been found to form a stable complex with horse heart cytochrome c that appears to be held together by electrostatic interactions. The stability of this complex and the sulfide:cytochrome c reductase activity of cytochrome c-552 are both ionic strength dependent, with maximal rates of cytochrome c reduction and extent of complex formation occurring over the same ionic strength range. Trifluoroacetylated cytochrome c is not reduced in the presence of cytochrome c-552 and sulfide, nor does it form a complex with cytochrome c-552. These results suggest the possible involvement of cytochrome c lysine residues in complex formation. Cytochrome c-552 migrates with an anomalously high apparent molecular weight on gel filtration columns equilibrated with low ionic strength buffers, suggesting the possibility of conformational changes or dimerization of the protein. However, complexation of cytochrome c-552 with cytochrome c still occurs at low ionic strength.     

A comparative study of EPR spin trapping and cytochrome c reduction techniques for the measurement of superoxide anions

Superoxide anions (O₂^.−) generated by the reaction of xanthine with xanthine oxidase were measured by the reduction of cytochrome c and by electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the relative sensitivities of these two techniques for the measurement of O₂^.−. Mixtures of xanthine, xanthine oxidase, DMPO generated two adducts, a transient DMPO-OOH and a smaller but longer-lived DMPO-OH. Both adducts were inhibited by superoxide dismutase (SOD), demonstrating they originated from O₂^.−, and were also significantly decreased when the experiments were performed using unchelated buffers, suggesting that metal ion impurities in unchelated buffers alter the formation or degradation of DMPO-adducts. O₂^.−, generated by concentrations of xanthine as low as 0.05 μM, were detectable using EPR spin trapping. In contrast, mixtures of xanthine, xanthine oxidase, and cytochrome c measured spectrophotometrically at 550 nm demonstrated that concentrations of xanthine above 1 μM were required to produce measurable levels of reduced cytochrome c. These studies demonstrate that spin trapping using DMPO was at least 20-fold more sensitive than the reduction of cytochrome c for the measurement of superoxide anions. However, at levels of superoxide generation where cytochrome c provides a linear measurement of production, EPR spin trapping may underestimate radical production, probably due to degradation of DMPO radical adducts.     

Molecular dynamics in cytochrome c oxidase Mössbauer spectra deconvolution

In this work low temperature molecular dynamics simulations of cytochrome c oxidase are used to predict an experimentally observable, namely Mössbauer spectra width. Predicted lineshapes are used to model Lorentzian doublets, with which published cytochrome c oxidase Mössbauer spectra were simulated. Molecular dynamics imposed constraints to spectral lineshapes permit to obtain useful information, like the presence of multiple chemical species in the binuclear center of cytochrome c oxidase. Moreover, a benchmark of quality for molecular dynamic simulations can be obtained. Despite the overwhelming importance of dynamics in electron–proton transfer systems, limited work has been devoted to unravel how much realistic are molecular dynamics simulations results. In this work, molecular dynamics based predictions are found to be in good agreement with published experimental spectra, showing that we can confidently rely on actual simulations. Molecular dynamics based deconvolution of Mössbauer spectra will lead to a renewed interest for application of this approach in bioenergetics.     

Characterization of purified cytochrome c1 from Rhodobacter sphaeroides R-26

Cytochrome c₁ from a photosynthetic bacterium Rhodobacter sphaeroides R-26 has been purified to homogeneity. The purified protein contains 30 nmol heme per mg protein, has an isoelectric point of 5.7, and is soluble in aqueous solution in the absence of detergents. The apparent molecular weight of this protein is about 150 000, determined by Bio Gel A-0.5 m column chromatography; a minimum molecular weight of 30 000 is obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The absorption spectrum of this cytochrome is similar to that of mammalian cytochrome c₁, but the amino acid composition and circular dichroism spectral characteristics are different. The heme moiety of cytochrome c₁ is more exposed than is that of mammalian cytochrome c₁, but less exposed than that of cytochrome c₂. Ferricytochrome c₁ undergoes photoreduction upon illumination with light under anaerobic conditions. Such photoreduction is completely abolished when p-chloromercuriphenylsulfonate is added to ferricytochrome c₁, suggesting that the sulfhydryl groups of cytochrome c₁ are the electron donors for photoreduction. Purified cytochrome c₁ contains 3 ± 0.1 mol of the p-chloromercuriphenylsulfonate titratable sulfhydryl groups per mol of protein. In contrast to mammalian cytochrome c₁, the bacterial protein does not form a stable complex with cytochrome c₂ or with mammalian cytochrome c at low ionic strength. Electron transfer between bacterial ferrocytochrome c₁ and bacterial ferricytochrome c₂, and between bacterial ferrocytochrome c₁ and mammalian ferricytochrome c proceeds rapidly with equilibrium constants of 49 and 3.5, respectively. The midpoint potential of purified cytochrome c₁ is calculated to be 228 mV, which is identical to that of mammalian cytochrome c₁.     

The distance between cytochromes a and a3 in the azide compound of bovine-heart cytochrome oxidase

The electron-spin relaxation rates of the two species of cytochrome a³⁺₃-azide found in the azide compound of bovine-heart cytochrome oxidase were measured by progressive microwave saturation at T = 10 K. It has been shown previously that Cyt a⁺³₃-azide gives rise to two distinct EPR resonances, depending upon the oxidation state of Cyt a. When Cyt a is ferrous, Cyt a³⁺₃-azide has g = 2.88, 2.19 and 1.64; upon oxidation of Cyt a, the a³⁺₃-azide g-values become g = 2.77, 2.18, and 1.74 (Goodman, G. (1984) J. Biol. Chem. 259, 15094–15099). The relaxation effect of Cyt a on Cyt a₃ could be measured as the difference in microwave field saturation parameter H_1/2 between the g = 2.77 and g = 2.88 species. For each signal the spin-lattice relaxation time T₁ was determined from H_1/2 using the transverse relaxation time T₂. The value of T₂ at 10 K was extrapolated from a plot of line-width vs. temperature at higher temperature. The dipolar contribution to T₁ was related to the Cyt a-Cyt a₃ spin-spin distance utilizing available information on the relative orientation of Cyt a₃-azide and Cyt a (Erecinska, M., Wilson, D.F. and Blasie, J.K. (1979) Biochim. Biophys. Acta 545, 352–364). By taking into account the relaxation parameters for both g_x and g_z components of the Cyt a₃-azide g-tensor, the angle between the g_z components of the Cyt a and Cyt a₃g-tensors was determined to be between 0 and 18°, and the Cyt a-Cyt a₃ spin-spin distance was found to be 19 ± 8 Å.     

Energy diagrams and mechanism for proton pumping in cytochrome c oxidase

The powerful technique of energy diagrams has been used to analyze the mechanism for proton pumping in cytochrome c oxidase. Energy levels and barriers are derived starting out from recent kinetic experiments for the O to E transition, and are then refined using general criteria and a few additional experimental facts. Both allowed and non-allowed pathways are obtained in this way. A useful requirement is that the forward and backward rate should approach each other for the full membrane gradient. A key finding is that an electron on heme a (or the binuclear center) must have a significant lowering effect on the barrier for proton uptake, in order to prevent backflow from the pump-site to the N-side. While there is no structural gating in the present mechanism, there is thus an electronic gating provided by the electron on heme a. A quantitative analysis of the energy levels in the diagrams, leads to Prop-A of heme a₃ as the most likely position for the pump-site, and the Glu278 region as the place for the transition state for proton uptake. Variations of key redox potentials and pK_a values during the pumping process are derived for comparison to experiments.     

Modulation of cytochrome oxidase kinetics by indirect antibody action

Polyclonal antibodies raised against isolated subunit V from beef heart cytochrome oxidase or against the intact enzyme increase its apparent affinity for the substrate cytochrome c at the high-affinity site while diminishing the turnover at that site. At the low-affinity site the major action of both types of antibody is to reduce the apparent affinity for cytochrome c. At high ionic strengths the kinetic effect of anti-subunit V is very small although it still binds to the enzyme. The results are interpreted in terms of a model for the enzyme in which antibodies can modulate cytochrome oxidase kinetics by affecting the binding of cytochrome c, even if the antibody-binding site is on a subunit not directly involved in substrate binding.     

Cytochrome c1 and b-type cytochrome with two heme centers in the photosynthetic membranes from Rhodopseudomonas palustris

The photosynthetic membranes from Rhodopseudomonas palustris contained one species of membrane-bound c-type cytochrome, presumably cytochrome c₁, and a b-type cytochrome with two heme centers. The molecular weight and midpoint potential of cytochrome c₁ were 30000 and 275 mV, respectively. The peak of the reduced-minus-oxidized difference spectrum of cytochrome c₁ was at 552 nm. Molecular weight of the b-type cytochrome was 32000 and the cytochrome had two midpoint potentials of 60 mV and −55 mV. The peaks of the reduced-minus-oxidized difference spectra of the high and low midpoint potential heme centers were at 560 and 562 nm, respectively. These results suggested that there was a cytochrome b-c₁ complex in Rps. palustris.