Direct measurement of proton release by cytochrome
            <i>c</i>
            oxidase in solution during the F→O transition

Zaslavsky, Dmitry; Sadoski, Robert C.; Rajagukguk, Sany; Geren, Lois; Millett, Francis; Durham, Bill; Gennis, Robert B.

doi:10.1073/pnas.0401521101

Cited by 24 publications

(24 citation statements)

References 32 publications

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“…The results from a recent study showed that in the monomeric form of the mitochondrial enzyme proton uptake during the F 3 3 O 4 transition is delayed such that (in H 2 O) proton release is observed before the proton uptake (31). This order of reactions is the reverse compared with that observed in the present study with R. sphaeroides CcO.…”

Section: Discussioncontrasting

confidence: 32%

“…However, it should be noted that in monomeric mitochondrial CcO the pH titration of the F 3 3 O 4 transition rate is shifted to higher pH values as compared with the dimeric form of the enzyme (49). This result indicates that around pH 7 proton uptake is not rate-limiting for the F 3 3 O 4 transition and that there is an internal proton pool in the monomeric enzyme (31). Thus, in contrast to the R. sphaeroides CcO, in the monomeric mitochondrial CcO the pumped proton is present within the enzyme already before initiation of the F 3 3 O 4 reaction.…”

Section: Discussionmentioning

confidence: 73%

“…Electron [29][30][31]. The different KIEs indicate that different proton-transfer events are rate-limiting for these reactions.…”

mentioning

confidence: 99%

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The timing of proton migration in membrane-reconstituted cytochrome c oxidase

Salomonsson

Faxén

Ädelroth

et al. 2005

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

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In mitochondria and aerobic bacteria energy conservation involves electron transfer through a number of membrane-bound protein complexes to O2. The reduction of O2, accompanied by the uptake of substrate protons to form H2O, is catalyzed by cytochrome c oxidase (CcO). This reaction is coupled to proton translocation (pumping) across the membrane such that each electron transfer to the catalytic site is linked to the uptake of two protons from one side and the release of one proton to the other side of the membrane. To address the mechanism of vectorial proton translocation, in this study we have investigated the solvent deuterium isotope effect of proton-transfer rates in CcO oriented in small unilamellar vesicles. Although in H2O the uptake and release reactions occur with the same rates, in D2O the substrate and pumped protons are taken up first (D Х 200 s, ''peroxy'' to ''ferryl'' transition) followed by a significantly slower proton release to the other side of the membrane (D Х 1 ms). Thus, the results define the order and timing of the proton transfers during a pumping cycle. Furthermore, the results indicate that during CcO turnover internal electron transfer to the catalytic site is controlled by the release of the pumped proton, which suggests a mechanism by which CcO orchestrates a tight coupling between electron transfer and proton translocation.heme-copper oxidases ͉ electron transfer ͉ membrane protein ͉ respiratory chain I n membrane-bound proton pumps the endergonic proton translocation across the membrane is driven by an exergonic reaction, e.g., electron transfer from a low-potential donor to a high-potential acceptor. The protons are translocated through proton-transfer pathways, typically composed of networks of hydrogen-bonded protonatable and polar amino acid side chains and bound water molecules, that span the transmembrane part of the protein. To prevent short-circuiting of the electrochemical gradient it is crucial that there is never simultaneous access (i.e., a direct contact) to the two membrane sides. One way to control the proton access is to alter the position of e.g., an amino acid side chain such that the group exchanges protons rapidly with either one side or the other side of the membrane (but never both sides simultaneously), defining an input and an output state (for review, see refs. 1-6). Proton pumping would be accomplished, for example, if the reaction that drives proton translocation is energetically coupled to changes in the pK a value of the protonatable group such that it has a high pK a in the input state and a low pK a in the output state. In the discussion below, we will refer to such a group as a ''pumping element.'' Cytochrome c oxidase (CcO), which belongs to the hemecopper oxidase superfamily, is an example of a redox-driven proton pump (for review, see refs 4, 5, and 7-12). The CcOs are found in the cytoplasmic membrane in bacteria or the inner membrane of mitochondria, where they catalyze the reduction of molecular oxygen to water. The electrons are donated by...

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Section: Discussioncontrasting

confidence: 32%

Section: Discussionmentioning

confidence: 73%

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The timing of proton migration in membrane-reconstituted cytochrome c oxidase

Salomonsson

Faxén

Ädelroth

et al. 2005

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

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“…This initial ET step is followed by rapid electron distribution between Cu A and heme a (29 -31). The described sequence of ET steps is true not only for fully oxidized CcO but also for the "peroxy" (P) and ferryl (F) forms of oxidase (30,31). In P and F, the reduction potential of the catalytic site has been estimated to be close to ϩ1.0 V (32), and therefore this site is the thermodynamically favored acceptor.…”

Section: Discussionmentioning

confidence: 98%

“…In P and F, the reduction potential of the catalytic site has been estimated to be close to ϩ1.0 V (32), and therefore this site is the thermodynamically favored acceptor. However, despite the favorable reduction potential, ET to the catalytic site occurs by the intramolecular transfer from heme a to heme a 3 (30,31).…”

Section: Discussionmentioning

confidence: 99%

Filling the Catalytic Site of Cytochrome c Oxidase with Electrons

Jancura

Antalı́k

Berka

et al. 2006

Journal of Biological Chemistry

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In the reductive phase of its catalytic cycle, cytochrome c oxidase receives electrons from external electron donors. Two electrons have to be transferred into the catalytic center, composed of heme a 3 and Cu B , before reaction with oxygen takes place. In addition, this phase of catalysis appears to be involved in proton translocation. Here, we report for the first time the kinetics of electron transfer to both heme a 3 and Cu B during the transition from the oxidized to the fully reduced state. The state of reduction of both heme a 3 and Cu B was monitored by a combination of EPR spectroscopy, the rapid freeze procedure, and the stoppedflow method. The kinetics of cytochrome c oxidase reduction by hexaamineruthenium under anaerobic conditions revealed that the rate-limiting step is the initial electron transfer to the catalytic site that proceeds with apparently identical rates to both heme a 3 and Cu B . After Cu B is reduced, electron transfer to oxidized heme a 3 is enhanced relative to the rate of entry of the first electron.Mitochondrial cytochrome c oxidase (CcO) 4 is a membrane protein that catalyzes the oxidation of ferrocytochrome c by molecular oxygen. The reduction of oxygen to water requires the delivery of four electrons and four protons into the catalytic center of the enzyme. Electrons enter oxidase from the cytosolic domain and protons from the matrix side of the inner mitochondrial membrane. This redox reaction is coupled to the pumping of four additional protons across the membrane. Both of these processes contribute to the generation of a transmembrane proton gradient.Bovine heart CcO contains 13 polypeptides; however, four redox centers involved in electron transport (ET) and in the reduction of O 2 to water are located in two subunits (1). Three of these centers, heme a, heme a 3 , and copper ion called Cu B , are in subunit I, and the dinuclear copper center Cu A is located in subunit II (1).Cu A is close to the cytosolic surface of the protein and serves as the acceptor of electrons either from the physiological reductant, ferrocytochrome c, or artificial electron donors (2-6). Electrons received by the oxidase are rapidly distributed between Cu A and heme a on the microsecond time scale (3, 4, 6 -9). ET continues further to the catalytic center composed of heme a 3 and Cu B . At this catalytic center, the interaction of electrons, protons, and oxygen occurs. The reaction of CcO with oxygen, however, can take place only when two electrons have reduced the binuclear center. This part of the catalytic cycle of CcO, during which electrons are delivered to CcO prior to reaction with oxygen, is usually referred to as the reductive phase. It appears that the reductive phase consists not only of ET reactions but is accompanied also by proton pumping (10). Despite the significance of these ET reactions, there is virtually no data on how electrons are delivered into the catalytic site and how this ET is affected by the presumed redox interaction between heme a 3 and Cu B .Under anaerobic condi...

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