Redox-driven proton pumping by heme-copper oxidases

Brzezinski, Peter; Larsson, Gisela

doi:10.1016/s0005-2728(03)00079-3

Cited by 140 publications

(139 citation statements)

References 84 publications

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“…Similar conclusions were found in the MCCE analysis of the P. denitrificans structure, where only 1 proton is also taken up on formation of the R (OORR) state (33). Only 1 proton is needed for the O-O bond splitting chemistry of bound O 2 in the A to P R transition (29,110,111). This proton and an electron are proposed to be donated by Tyr I-288, forming a hydroxyl on Cu B and unprotonated heme a 3 ferryl species (Fe 4+ dO 2-) (75,(92)(93)(94)(95)(96).…”

Section: Discussionsupporting

confidence: 79%

Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

2006

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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 ∆pK 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 ≈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.Heme-copper oxidases are the terminal electron acceptors in anaerobic organisms. These transmembrane proteins reduce dioxygen and convert the released chemical energy into an electrochemical gradient, across the eukaryotic mitochondrial membrane or the bacterial cell membrane (1-4). Cytochrome c oxidases are the most prevalent hemecopper oxidases. In this protein 4 electrons, provided by 4 cytochromes c, are used to reduce dioxygen to water. The 4 protons needed to make water are taken from the negative cytoplasmic side of the membrane, adding to the electrochemical gradient. Four additional protons are pumped across the membrane (5). Thus, each dioxygen molecule reduced by cytochrome c oxidase is coupled to the transfer of 8 charges across the membrane.The first step of electron transfer is from cytochrome c to Cu A , a dicopper center in subunit II which extends beyond the membrane on the proton release side of the protein (Figure 1). After receiving the electron, Cu A reduces the 6-coordinate, low-spin, bis-Hi...

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Section: Discussionsupporting

confidence: 79%

Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

2006

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“…2. This is the picture that emerged from an instructive recent study of the O to E step by Wikström and coworkers (15,21), where some points about the proton donor and acceptor confirm the proposal of Brzezinski and coworkers (4) and also coincide with our recent theoretical and conceptual analyses (17). In this picture, the primary PT is triggered by ET to heme a, and it occurs most probably by transferring a proton from E286 to Prd.…”

Section: Functional Constraintssupporting

confidence: 83%

Electrostatic basis for the unidirectionality of the primary proton transfer in cytochrome c oxidase

Pisliakov

Sharma

Chu

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

123

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Gaining detailed understanding of the energetics of the protonpumping process in cytochrome c oxidase (CcO) is one of the challenges of modern biophysics. Despite promising mechanistic proposals, most works have not related the activation barriers of the different assumed steps to the protein structure, and there has not been a physically consistent model that reproduced the barriers needed to create a working pump. This work reevaluates the activation barriers for the primary proton transfer (PT) steps by calculations that reflect all relevant free energy contributions, including the electrostatic energies of the generated charges, the energies of water insertion, and large structural rearrangements of the donor and acceptor. The calculations have reproduced barriers that account for the directionality and sequence of events in the primary PT in CcO. It has also been found that the PT from Glu-286 (E) to the propionate of heme a3 (Prd) provides a gate for an initial back leakage from the high pH side of the membrane. Interestingly, the rotation of E that brings it closer to Prd appears to provide a way for blocking competing pathways in the primary PT. Our study elucidates and quantifies the nature of the control of the directionality in the primary PT in CcO and provides instructive insight into the role of the water molecules in biological PT, showing that ''bridges'' of several water molecules in hydrophobic regions present a problem (rather than a solution) that is minimized in the primary PT.coupled electron transfer proton transfer ͉ proton pumps ͉ dielectric effects ͉ free energy calculations ͉ pK a in nonpolar regions

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“…The scenario outlined in this section was previously summarized in the context of a molecular mechanism for proton pumping by the heme-copper oxidases (46), which is shown in Fig. 5 also to visualize the data obtained from the present study.…”

Section: Discussionmentioning

confidence: 92%

Controlled uncoupling and recoupling of proton pumping in cytochrome c oxidase

Brändén

Pawate

Gennis

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain and couples energetically the reduction of oxygen to water to proton pumping across the membrane. The results from previous studies showed that proton pumping can be uncoupled from the O 2-reduction reaction by replacement of one single residue, Asn-139 by Asp (N139D), located Ϸ30 Å from the catalytic site, in the D-proton pathway. The uncoupling was correlated with an increase in the pK a of an internal proton donor, Glu-286, from Ϸ9.4 to >11. Here, we show that replacement of the acidic residue, Asp-132 by Asn in the N139D CcO (D132N͞N139D double-mutant CcO) results in restoration of the Glu-286 pK a to the original value and recoupling of the proton pump during steady-state turnover. Furthermore, a kinetic investigation of the specific reaction steps in the D132N͞N139D double-mutant CcO showed that proton pumping is sustained even if proton uptake from solution, through the D-pathway, is slowed. However, during single-turnover oxidation of the fully reduced CcO the P 3 F transition, which does not involve electron transfer to the catalytic site, was not coupled to proton pumping. The results provide insights into the mechanism of proton pumping by CcO and the structural elements involved in this process.cytochrome oxidase ͉ electron transfer ͉ gating ͉ proton transfer

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Redox-driven proton pumping by heme-copper oxidases

Cited by 140 publications

References 84 publications

Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

Electrostatic basis for the unidirectionality of the primary proton transfer in cytochrome c oxidase

Controlled uncoupling and recoupling of proton pumping in cytochrome c oxidase

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