Uptake and release of protons during the reaction between cytochrome c oxidase and molecular oxygen: a flow-flash investigation

Oliveberg, Mikael; Hallén, Stefan; Nilsson, T.

doi:10.1021/bi00216a019

Cited by 87 publications

(78 citation statements)

References 13 publications

(24 reference statements)

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“…It has been reported that only one proton equivalent per fully reduced enzyme is pumped upon complete oxidation of the fully reduced enzyme with O 2 (14,15). (16).…”

Section: Resultsmentioning

confidence: 99%

The low-spin heme of cytochrome c oxidase as the driving element of the proton-pumping process

Tsukihara

Shimokata

Katayama

et al. 2003

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

402

467

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Mitochondrial cytochrome c oxidase plays an essential role in aerobic cellular respiration, reducing dioxygen to water in a process coupled with the pumping of protons across the mitochondrial inner membrane. An aspartate residue, Asp-51, located near the enzyme surface, undergoes a redox-coupled x-ray structural change, which is suggestive of a role for this residue in redoxdriven proton pumping. However, functional or mechanistic evidence for the involvement of this residue in proton pumping has not yet been obtained. We report that the Asp-51 3 Asn mutation of the bovine enzyme abolishes its proton-pumping function without impairment of the dioxygen reduction activity. Improved x-ray structures (at 1.8͞1.9-Å resolution in the fully oxidized͞ reduced states) show that the net positive charge created upon oxidation of the low-spin heme of the enzyme drives the active proton transport from the interior of the mitochondria to Asp-51 across the enzyme via a water channel and a hydrogen-bond network, located in tandem, and that the enzyme reduction induces proton ejection from the aspartate to the mitochondrial exterior. A peptide bond in the hydrogen-bond network critically inhibits reverse proton transfer through the network. A redoxcoupled change in the capacity of the water channel, induced by the hydroxyfarnesylethyl group of the low-spin heme, suggests that the channel functions as an effective proton-collecting region. Infrared results indicate that the conformation of Asp-51 is controlled only by the oxidation state of the low-spin heme. These results indicate that the low-spin heme drives the proton-pumping process.

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“…It has been reported that only one proton equivalent per fully reduced enzyme is pumped upon complete oxidation of the fully reduced enzyme with O 2 (14,15). (16).…”

Section: Resultsmentioning

confidence: 99%

The low-spin heme of cytochrome c oxidase as the driving element of the proton-pumping process

Tsukihara

Shimokata

Katayama

et al. 2003

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

402

467

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“…2) [11,12,15,46]. The role of the redox-linked movement of D51 is also compatible with proton release upon oxidation of the metal centers.…”

Section: A Cooperative Proton Pump and Role Of Heme A In Cytochrome Cmentioning

confidence: 94%

A cooperative model for protonmotive heme‐copper oxidases. The role of heme a in the proton pump of cytochrome c oxidase

Papa¹,

Capitanio²,

Villani³

1998

FEBS Letters

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Oxido-reductions of metal centers in cytochrome c oxidase are linked to pK shifts of acidic groups in the enzyme (redox Bohr effects). The linkage at heme a results in proton uptake from the inner space upon reduction and proton release in the external space upon oxidation of the metal. The relationship of this process to the features of the proton pump in cytochrome c oxidase and its atomic structure revealed by X-ray crystallography to 2.8^2.3 A î resolution is examined. A mechanism for the proton pump of cytochrome c oxidase, based on cooperative coupling at heme a, is proposed.z 1998 Federation of European Biochemical Societies.

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“…The conversion of oxidized CcO to the MV-CO derivative seems to be accompanied by the uptake of two protons (M.F. and G.P., unpublished work), whereas the conversion of MV-CO to the P form occurs without any demonstrable change in pH (37). Thus, there are sufficient protons sequestered in the enzyme to support formation of either OH Ϫ or of H 2 O.…”

mentioning

confidence: 97%

Mass spectrometric determination of dioxygen bond splitting in the “peroxy” intermediate of cytochrome c oxidase

Fabián

Wong

Gennis

et al. 1999

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

122

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The ''peroxy'' intermediate (P form) of bovine cytochrome c oxidase was prepared by reaction of the two-electron reduced mixedvalence CO complex with 18 O2 after photolytic removal of CO. The water present in the reaction mixture was recovered and analyzed for 18 O enrichment by mass spectrometry. It was found that approximately one oxygen atom ( 18 O) per one equivalent of the P form was present in the bulk water. The data show that the oxygen-oxygen dioxygen bond is already broken in the P intermediate and that one oxygen atom can be readily released or exchanged with the oxygen of the solvent water. Bovine cytochrome c oxidase (CcO) catalyzes the oxidation of ferrocytochrome c and the reduction of dioxygen to water in a process that conserves a fraction of the energy released in the redox process by translocating protons across the inner membrane of the mitochondrion. For this process, the enzyme depends on four transition metal centers: cytochrome a, Cu A , cytochrome a 3 , and Cu B . Electrons from ferrocytochrome c are transferred to Cu A and subsequently delivered to the cytochrome a 3 -Cu B binuclear center via cytochrome a; the conversion of O 2 to H 2 O is effected at this binuclear center.When the enzyme is fully reduced (Fe ϩ2 a Cu ϩ A Fe ϩ2 a3 Cu ϩ B ), a process requiring four electrons, the reaction with O 2 is fast, and detection of intermediates usually requires rapid kinetic techniques and transient spectroscopic methods. In contrast, when partially reduced oxidase reacts with O 2 , it is possible to obtain relatively stable oxygenated intermediates of the enzyme (1, 2). One of these intermediates, the ''peroxy'' or P form, is a product of the reaction of the two-electron reduced enzyme with O 2 . This P form is readily prepared on reaction of the two-electron reduced mixed-valence CO complex (MV-CO or Fe ϩ3 a Cu ϩ2 A Fe ϩ2 a3 CO Cu ϩ B ) with oxygen after photolytic removal of the bound CO (3-9). The same compound P can be observed after reaction of oxidized enzyme with hydrogen peroxide (10-14) and in mitochondria during reverse electron flow (15-17). Because the P form of the enzyme is formally at the two-electron reduced state, it was initially expected to have an intact oxygen-oxygen bond (e.g., Fe refs. 10, 12, 15, and 17-19). However, recent resonance Raman experiments (20-23) and data from reductive titrations of the P form (24) suggest that the O-O bond is broken, and the presence of H 2 O 2 in this species could not be detected (25).Cleavage of the oxygen-oxygen bond requires that an additional two reducing equivalents be available. These would need to be provided by one or more groups present in the enzyme. The reaction mechanism might then be analogous to the formation of compound I in heme peroxidases, during which reaction hydrogen peroxide is decomposed.Because the nature of the P intermediate of CcO is controversial and our understanding of it is based entirely on spectroscopic observations, we have addressed the question directly as to whether the dioxygen bond is broken in thi...

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Uptake and release of protons during the reaction between cytochrome c oxidase and molecular oxygen: a flow-flash investigation

Cited by 87 publications

References 13 publications

The low-spin heme of cytochrome c oxidase as the driving element of the proton-pumping process

The low-spin heme of cytochrome c oxidase as the driving element of the proton-pumping process

A cooperative model for protonmotive heme‐copper oxidases. The role of heme a in the proton pump of cytochrome c oxidase

Mass spectrometric determination of dioxygen bond splitting in the “peroxy” intermediate of cytochrome c oxidase

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