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Calculated Proton Uptake on Anaerobic Reduction of Cytochrome c Oxidase:  Is the Reaction Electroneutral?

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journal contribution
posted on 04.07.2006, 00:00 by Yifan Song, Ekaterina Michonova-Alexova, M. R. Gunner
Cytochrome c oxidase is a transmembrane proton pump that builds an electrochemical gradient using chemical energy from the reduction of O2. Ionization states of all residues were calculated with Multi-Conformation Continuum Electrostatics (MCCE) in seven anaerobic oxidase redox states ranging from fully oxidized to fully reduced. One long-standing problem is how proton uptake is coupled to the reduction of the active site binuclear center (BNC). The BNC has two cofactors:  heme a3 and CuB. If the protein needs to maintain electroneutrality, then 2 protons will be bound when the BNC is reduced by 2 electrons in the reductive half of the reaction cycle. The effective pKas of ionizable residues around the BNC are evaluated in Rhodobacter sphaeroides cytochrome c oxidase. At pH 7, only a hydroxide coordinated to CuB shifts its pKa from below 7 to above 7 and so picks up a proton when heme a3 and CuB are reduced. Glu I-286, Tyr I-288, His I-334, and a second hydroxide on heme a3 all have pKas above 7 in all redox states, although they have only 1.6−3.5 ΔpK units energy cost for deprotonation. Thus, at equilibrium, they are protonated and cannot serve as proton acceptors. The propionic acids near the BNC are deprotonated with pKas well below 7. They are well stabilized in their anionic state and do not bind a proton upon BNC reduction. This suggests that electroneutrality in the BNC is not maintained during the anaerobic reduction. Proton uptake on reduction of CuA, heme a, heme a3, and CuB shows ≈2.5 protons bound per 4 electrons, in agreement with prior experiments. One proton is bound by a hydroxyl group in the BNC and the rest to groups far from the BNC. The electrochemical midpoint potential (Em) of heme a is calculated in the fully oxidized protein and with 1 or 2 electrons in the BNC. The Em of heme a shifts down when the BNC is reduced, which agrees with prior experiments. If the BNC reduction is electroneutral, then the heme a Em is independent of the BNC redox state.