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Net proton uptake is preceded by multiple proton transfer steps upon electron injection into cytochrome c oxidase.

ABSTRACT: Cytochrome c oxidase (COX), the last enzyme of the respiratory chain of aerobic organisms, catalyzes the reduction of molecular oxygen to water. It is a redox-linked proton pump, whose mechanism of proton pumping has been controversially discussed, and the coupling of proton and electron transfer is still not understood. Here, we investigated the kinetics of proton transfer reactions following the injection of a single electron into the fully oxidized enzyme and its transfer to the hemes using time-resolved absorption spectroscopy and pH indicator dyes. By comparison of proton uptake and release kinetics observed for solubilized COX and COX-containing liposomes, we conclude that the 1-?s electron injection into Cu(A), close to the positive membrane side (P-side) of the enzyme, already results in proton uptake from both the P-side and the N (negative)-side (1.5 H(+)/COX and 1 H(+)/COX, respectively). The subsequent 10-?s transfer of the electron to heme a is accompanied by the release of 1 proton from the P-side to the aqueous bulk phase, leaving ?0.5 H(+)/COX at this side to electrostatically compensate the charge of the electron. With ?200 ?s, all but 0.4 H(+) at the N-side are released to the bulk phase, and the remaining proton is transferred toward the hemes to a so-called "pump site." Thus, this proton may already be taken up by the enzyme as early as during the first electron transfer to Cu(A). These results support the idea of a proton-collecting antenna, switched on by electron injection.

SUBMITTER: Kirchberg K

PROVIDER: S-EPMC3318725 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Net proton uptake is preceded by multiple proton transfer steps upon electron injection into cytochrome c oxidase.

Kirchberg Kristina K Michel Hartmut H Alexiev Ulrike U

The Journal of biological chemistry 20120111 11

Cytochrome c oxidase (COX), the last enzyme of the respiratory chain of aerobic organisms, catalyzes the reduction of molecular oxygen to water. It is a redox-linked proton pump, whose mechanism of proton pumping has been controversially discussed, and the coupling of proton and electron transfer is still not understood. Here, we investigated the kinetics of proton transfer reactions following the injection of a single electron into the fully oxidized enzyme and its transfer to the hemes using t ...[more]

PMID: 22238345

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Project description:Cytochrome c oxidase is a transmembrane proton pump that builds an electrochemical gradient using chemical energy from the reduction of O(2). 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 a(3) and Cu(B). 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 pK(a)s of ionizable residues around the BNC are evaluated in Rhodobacter sphaeroides cytochrome c oxidase. At pH 7, only a hydroxide coordinated to Cu(B) shifts its pK(a) from below 7 to above 7 and so picks up a proton when heme a(3) and Cu(B) are reduced. Glu I-286, Tyr I-288, His I-334, and a second hydroxide on heme a(3) all have pK(a)s above 7 in all redox states, although they have only 1.6-3.5 DeltapK 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 pK(a)s 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 Cu(A), heme a, heme a(3), and Cu(B) shows approximately 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 (E(m)) of heme a is calculated in the fully oxidized protein and with 1 or 2 electrons in the BNC. The E(m) 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 E(m) is independent of the BNC redox state.

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Net proton uptake is preceded by multiple proton transfer steps upon electron injection into cytochrome c oxidase.

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Net proton uptake is preceded by multiple proton transfer steps upon electron injection into cytochrome c oxidase.

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