Electron-transport-driven proton pumps display nonhyperbolic kinetics: Simulation of the steady-state kinetics of cytochrome
            <i>c</i>
            oxidase

Brzezinski, Peter; Malmström, Bo G.

doi:10.1073/pnas.83.12.4282

Cited by 48 publications

(20 citation statements)

References 23 publications

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“…The free energy barriers found in the MS-EVB simulations indicate a high pumping rate for this process, which is in agreement with the “kinetic gating” theory [37, 40]. Because the excess proton PMF is very flat for proton pumping, a mechanism to prevent proton back leak may be important.…”

Section: Resultssupporting

confidence: 80%

Expanding the view of proton pumping in cytochrome c oxidase through computer simulation

Peng

Voth

2012

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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In cytochrome c oxidase (CcO), a redox-driven proton pump, protons are transported by the Grotthuss shuttling via hydrogen-bonded water molecules and protonatable residues. Proton transport through the D-pathway is a complicated process that is highly sensitive to alterations in the amino acids or the solvation structure in the channel, both of which can inhibit proton pumping and enzymatic activity. Simulations of proton transport in the hydrophobic cavity showed a clear redox state dependence. To study the mechanism of proton pumping in CcO, multi-state empirical valence bond (MS-EVB) simulations have been conducted, focusing on the proton transport through the D-pathway and the hydrophobic cavity next to the binuclear center. The hydration structures, transport pathways, effects of residues, and free energy surfaces of proton transport were revealed in these MS-EVB simulations. The mechanistic insight gained from them is herein reviewed and placed in context for future studies.

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Section: Resultssupporting

confidence: 80%

Expanding the view of proton pumping in cytochrome c oxidase through computer simulation

Peng

Voth

2012

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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“…However, previous experiments carried out using carboxyl-modifying reagents indicate that several nuclearencoded subunits constitute part of the interaction domain in the mitochondrial oxidase (Bisson & Montecucco, 1982;Kadenbach & Stroh, 1984;Capaldi, 1990). In contrast to the intact oxidase, no biphasic behavior could be observed in any salt concentrations in the reaction between the isolated CUA domain and cytochrome c. This is in line with proposals of Antalis and Palmer (1 982) and Brzezinski and Malmstrom (1986) that the biphasic kinetics may arise from changes within the subunit I rather than from two distinct cytochrome c binding sites. However, our experiments do not exclude the presence of a second lowaffinity site constructed by proteins other than subunit I1 in the eukaryotic enzyme complex.…”

Section: Cytochrome C Binding Site Resides Within the Cua Domainsupporting

confidence: 89%

Electron Transfer between Cytochrome c and the Isolated CuA Domain: Identification of Substrate-Binding Residues in Cytochrome c Oxidase

Lappalainen

Watmough²,

Greenwood³

et al. 1995

Biochemistry

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Subunit II of cytochrome c oxidase has a C-terminal domain that is exposed to aqueous solution on membrane surface and contains a copper center called CuA. The central part of the cytochrome c binding site is thought to reside in this domain. We have expressed the subunit II fragment of the Paracoccus denitrificans cytochrome c oxidase in a soluble form and studied its interaction with cytochrome c by stopped-flow spectroscopy. The oxidation of cytochrome c by the CuA domain follows monophasic kinetics, indicating the presence of a single kinetically competent binding site. In low ionic strength medium, the domain oxidizes Paracoccus cytochrome c-550 and horse mitochondrial cytochrome c at the rates of 1.5 x 10(6) and 3 x 10(5) M-1 s-1, respectively. The reaction rates are strongly dependent on ionic strength, which must reflect electrostatic interactions within the complex. The KD for the complex between the bacterial cytochrome c and the domain is 1.6 microM; i.e., it is similar to that between the mitochondrial cytochrome c and the intact oxidase, suggesting that both contain the same catalytically competent binding site. Using site-directed mutagenesis, we have identified five conserved residues of the CuA domain that are involved in the cytochrome c binding. Mutations of glutamine 148, glutamate 154, aspartate 206, aspartate 221, or glutamate 246 lead to a 35-85% decrease in the rate of cytochrome c oxidation. The simultaneous substitution of three invariant carboxylic acids (aspartate 206, aspartate 221, and glutamate 246) leads to a 95% decrease in the reaction rate. Conversely, the reaction can be enhanced by removing a positive charge (lysine 219) from the CuA domain.

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“…To account for biphasic cytochrome c kinetics, Brzezinski and Malmstrom (1986) suggest that redox proton pumps have two oxidized forms, one which binds H + at one face and another which releases H + at a second face. Both forms react with cytochrome c, although the affinities differ.…”

Section: Discussionmentioning

confidence: 99%

Routes of electron transfer in beef heart cytochrome c oxidase: is there a unique pathway used by all reductants?

Crinson

Nicholls²

1992

Biochem. Cell Biol.

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Cytochrome c oxidase oxidizes several hydrogen donors, including TMPD (N,N,N',N'-tetramethyl-p-phenyl-enediamine) and DMPT (2-amino-6,7-dimethyl-5,6,7,8-tetrahydropterine), in the absence of the physiological substrate cytochrome c. Maximal enzyme turnovers with TMPD and DMPT alone are rather less than with cytochrome c, but much greater than previously reported if extrapolated to high reductant levels and (or) to 100% reduction of cytochrome a in the steady state. The presence of cytochrome c is, therefore, not necessary for substantial intramolecular electron transfer to occur in the oxidase. A direct bimolecular reduction of cytochrome a by TMPD is sufficient to account for the turnover of the enzyme. CuA may not be an essential component of the TMPD oxidase pathway. DMPT oxidation seems to occur more rapidly than the DMPT--cytochrome a reduction rate and may therefore imply mediation of CuA. Both "resting" and "pulsed" oxidases contain rapid-turnover and slow-turnover species, as determined by aerobic steady-state reduction of cytochrome a by TMPD. Only the "rapid" fraction (approximately 70% of the total with resting and approximately 85% of the total with pulsed) is involved in turnover. We conclude that electron transfer to the a3CuB binuclear centre can occur either from cytochrome a or CuA, depending upon the redox state of the binuclear centre. Under steady-state conditions, cytochrome a and CuA may not always be in rapid equilibrium. Rapid enzyme turnover by either natural or artificial substrates may require reduction of both and two pathways of electron transfer to the a3CuB centre.

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Electron-transport-driven proton pumps display nonhyperbolic kinetics: Simulation of the steady-state kinetics of cytochrome c oxidase

Cited by 48 publications

References 23 publications

Expanding the view of proton pumping in cytochrome c oxidase through computer simulation

Expanding the view of proton pumping in cytochrome c oxidase through computer simulation

Electron Transfer between Cytochrome c and the Isolated CuA Domain: Identification of Substrate-Binding Residues in Cytochrome c Oxidase

Routes of electron transfer in beef heart cytochrome c oxidase: is there a unique pathway used by all reductants?

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