Proton transfer from glutamate 286 determines the transition rates between oxygen intermediates in cytochrome c oxidase

Ädelroth, Pia; Karpefors, Martin; Gilderson, Gwen; Tomson, Farol L.; Gennis, Robert B.; Brzezinski, Peter

doi:10.1016/s0005-2728(00)00194-8

Cited by 70 publications

(79 citation statements)

References 21 publications

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“…The rate of the F 3 3 O 4 transition has been modeled as the fraction of the fourth electron residing at the catalytic site multiplied by the proton-transfer rate to the catalytic site (14,32,40). With the double-mutant CcO, the F 3 3 O 4 transition is biphasic where the fast phase ( Х 1 ms; relative amplitude 40%) displays a pH-independent rate in the range 6-10.5, which indicates that the proton is supplied by an internal donor.…”

Section: Discussionmentioning

confidence: 99%

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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Section: Discussionmentioning

confidence: 99%

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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“…The next step in the reaction is transfer of a chemical proton via the D-channel and protonation of OH À bound to Fe-a 3 . It results in the pumping of the third proton and generation of a stable O (in our notation H p ) state [25,29]. One proton is pumped in the F p to H p transition.…”

Section: ''Pmentioning

confidence: 99%

Proton pumping mechanism and catalytic cycle of cytochrome c oxidase: Coulomb pump model with kinetic gating

POPOVI

2004

FEBS Letters

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Using electrostatic calculations, we have examined the dependence of the protonation state of cytochrome c oxidase from bovine heart on its redox state. Based on these calculations, we propose a possible scheme of redox-linked proton pumping. The scheme involves His291 -one of the ligands of the Cu B redox center -which plays the role of the proton loading site (PLS) of the pump. The mechanism of pumping is based on ET reaction between two hemes of the enzyme, which is coupled to a transfer of two protons. Upon ET, the first proton (fast reaction) is transferred to the PLS (His291), while subsequent transfer of the second ''chemical'' proton to the binuclear center (slow reaction) is accompanied by the ejection of the first (pumped) proton. Within the proposed model, we discuss the catalytic cycle of the enzyme.

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“…The D channel begins with Asp I-132 near the protein surface and ends with Glu I-286 near the BNC (6)(7)(8). The K channel begins with Glu II-101 (9,10), passes Lys I-362 (11), and ends with Tyr I-288.…”

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confidence: 99%

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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Proton transfer from glutamate 286 determines the transition rates between oxygen intermediates in cytochrome c oxidase

Cited by 70 publications

References 21 publications

Controlled uncoupling and recoupling of proton pumping in cytochrome c oxidase

Controlled uncoupling and recoupling of proton pumping in cytochrome c oxidase

Proton pumping mechanism and catalytic cycle of cytochrome c oxidase: Coulomb pump model with kinetic gating

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

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