Roy Mitchell

Proton uptake by cytochrome c oxidase on reduction and on ligand binding

On reduction, cytochrome oxidase was found to take up 2.4 +/- 0.1 protons in the pH range 7.2-8.5, of which 2 are associated with the binuclear centre, and the remaining fractional proton with haem a/CuA. Ligation to oxidised cytochrome oxidase of the azide, formate, fluoride or cyanide anions is accompanied by uptake of one proton. In the case of the reduced enzyme, no protonation changes are observed on binding O2 (Hallén S. and Nilsson T. (1992) Biochemistry 31, 11853-11859) or CO. Cyanide binding to reduced oxidase is, in contrast, still accompanied by uptake of a proton. These findings are discussed in terms of our previously-published proposal for the ligand chemistry of the binuclear site. The results overall suggest a principle of electroneutrality of redox and ligand state changes of the binuclear centre, with charge compensations provided only by protonation reactions.

Chemiosmotic coupling in cytochrome oxidase. Possible protonmotive O loop and O cycle mechanisms.

Using the principle of specific vectorial ligand conduction, we outline directly coupled protonmotive O loop and O cycle mechanisms of cytochrome oxidase action that are analogous to protonmotive Q loop and Q cycle mechanisms of QH2 dehydrogenase action. We discuss these directly coupled mechanisms in the light of available experimental knowledge, and suggest that they may stimulate useful new research initiatives designed to elucidate the osmochemistry of protonmotive oxygen reduction in cytochrome oxidase.

Proton-translocating pyrophosphatase of Rhodospirillum rubrum.

The membrane of chromatophores of Rhodospirillum rubrum contains an oligomycin-insensitive reversible pyrophosphatase system [l] that is involved in energy transduction by a pathway separate from that of the oligomycin-sensitive ATPase system [2-61. Pyrophosphate hydrolysis by chromatophores induces a change of carotenoid absorbance [7], a change in the fluorescence of added 8-anilinonaphthalene-1-sulphonic acid [8] and an uptake of phenyl dicarbaundecaborane anion [9]. Therefore it has been inferred that pyrophosphate hydrolysis by the pyrophosphatase system is coupled to (electrogenic) ion translocation across the chromatophore membrane; but the species of ion involved has not yet been identified. In this paper we describe measurements of changes of the pH and pK of the outer medium (pH, and pK,) during the hydrolysis of pulses of inorganic pyrophosphate (PPi) by suspensions of chromatophores from R. rubrum under various conditions, and we show that the pyrophosphatase system translocates protons, as suggested earlier [ 10, 111.

Binuclear centre structure of terminal protonmotive oxidases

The recent proliferation of data obtained from mutant forms of cytochrome oxidase and analogous enzymes has necessitated a re‐examination of existing structural models. A new model is proposed, consistent with these data, which brings several protonatable residues (Y244, D298, D300, T309, T316, K319, T326) into the vicinity of the binuclear centre, suggestive of a proton‐transferring function. In addition, we also consider those residues which may participate in electron transport between CuA and haem a. We suggest several potential lines of investigation.

Protonation states of the catalytic intermediates of cytochrome c oxidase

Protonation changes accompanying conversion of oxidised (O state) cytochrome c oxidase to the 2-electron-reduced P state, and 3-electron-reduced F state at pH 8.0 have been measured. It was found that 2 and 3 protons, respectively, were taken up. The fourth proton required for the reduction of O2 to H2O must therefore be consumed in the remaining F----O portion of the catalytic cycle.

Coupling of charge and proton movement in cytochrome c oxidase

In order to understand the protonmotive chemistry of terminal oxidases, we have explored the question of the degree to which the net charge of the reaction cycle intermediates is counterbalanced by the uptake of protons. This possibility for charge compensation by protonation originates in free energy considerations and the factors that control redox potentials (for a review see Ref. [1]). One of these is the difference in electrostatic energy between a reduced (with one net charge) and oxidised (with zero net charge) species, which can be expressed by the equation:

The assignment of the 655 nm spectral band of cytochrome oxidase.

The spectral characteristics of the ‘655 nm’ band of cytochrome oxidase were found to be affected by ligands of the binuclear centre, including formate and chloride, and by the resting/pulsed transition. The band titrated with near n=1 characteristics at a midpoint of about 400 mV, in contrast to haem a 3, which exhibits strong redox interaction and a titration range at significantly lower potential. Thus, although the total reduced‐oxidised difference spectrum of haem a 3, shows a trough at about 655 nm, this characteristic is absent in the low potential region. The 655 nm feature may arise from a charge transfer band of ferric high‐spin haem a 3, which is modulated by the redox state of CuB, as suggested by Beinert et al. [(1976) Biochim. Biophys. Acta 423, 339–355].

[69] Measurement of →H+/O in mitochondria and submitochondrial vesicles

Publisher Summary Quantitative measurement of the translocation of protons through the cristae membrane of mitochondria during the activity of the respiratory chain has assumed increasing importance since it was realized that proton translocation is an intrinsic function of redox chain systems generally and that these systems catalyze chemiosmotic rather than purely chemical reactions. The →H+/O or →H+/2e− quotient characteristic of the whole respiratory chain, or of a given part of it, represents the stoichiometry of the relevant chemiosmotic reaction. Thus, the →H+/O or →H+/2e− quotient is an essential datum for exploring the arrangement and molecular mechanics of the electron-carrying and hydrogen-carrying components of the cytochrome system and of the other proton-motive complexes of the respiratory chain of mitochondria. The translocation of protons across the cristae membrane of mitochondria or submitochondrial vesicles involves the transfer of both acid equivalents and electric charge through the membrane from the aqueous phase on one side to that on the other. In suspensions of mitochondria or submitochondrial particles, only the continuous outer aqueous phase or suspension medium is accessible to electrode systems designed to monitor changes of pH and ionic composition. Advantage can be taken of the migration of the charge-neutralizing ion species to measure the total charge translocation associated with the translocation of acid equivalents.

Measurement of the proton-motive stoichiometry of the respiratory chain of rat liver mitochondria: the effect of N-ethylmaleimide.

The apparent proton-motive stoichiometry as measured by the oxygen-pulse technique in KCl medium is depressed by the rapid uptake of inorganic phosphate, unless endogenous phosphate is depleted or uptake is inhibited. In sucrose or choline chloride media, where the internal pH is more acid than in KCl media, uptake may be greatly diminished. In the absence of significant phosphate uptake, the observed stoichiometry of around 8, obtained with no added substrate or respiratory inhibitors, appears to be characteristic of NADH oxidation without significant participation of the proton-translocating NAD(P) transhydrogenase. A mechanistic stoichiometry of at least 8 is indicated.

Protonmotive stoichiometry of rat liver cytochrome c oxidase: determination by a new rate/pulse method.

The stoichoimetry of vectorial H+ ejection coupled to electron flow through the cytochrome c oxidase (EC 1.9.3.1) of rat liver mitochondria was determined by a new rate/pulse method. This is a modification of the oxygen-pulse method. Electron flow through the oxidase is initiated by adding oxygen to suspensions of anaerobic mitochondria at a known and constant rate. Cytochrome c oxidase was examined directly or in combination with cytochrome c reductase (ubiquinol:ferricytochrome c oxidoreductase). In both cases the----H0+/2e- ratio was found to be constant during the time-course of oxygen reduction, and thus independent of delta pH. The stoichiometries observed were consistent with mechanistic stoichiometries of 2 and 6 for cytochrome c oxidase alone and cytochrome c oxidase together with cytochrome c reductase, respectively. The stoichiometry of cytochrome c reductase alone was also examined, by using ferricyanide in place of oxygen. The results obtained were consistent with the accepted mechanistic stoichiometry of 4 for this enzyme.