The proton pumping pathway of bovine heart cytochrome
            <i>c</i>
            oxidase

Shimokata, Kunitoshi; Katayama, Yukie; Murayama, Haruka; Suematsu, Makoto; Tsukihara, Tomitake; Muramoto, Kazumasa; Aoyama, Hiroshi; Yoshikawa, Shinya; Shimada, Hideo

doi:10.1073/pnas.0611627104

Cited by 133 publications

(142 citation statements)

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“…For elucidation of the structural bases for the mechanism of the proton collection and timely closure of the water channel, conformational dynamics after photolysis of CO (an O 2 analog)-bound CcO was examined using a newly developed time-resolved infrared system feasible for accurate detection of a single C‫؍‬O stretch band of ␣-helices of CcO in H 2 O medium. The present results indicate that migration of CO from heme a 3 Cytochrome c oxidase (CcO), 4 the terminal oxidase of cellular respiration, reduces O 2 to H 2 O. This process occurs at a site that includes an iron site (Fe a3 of heme a 3 ) and a copper site (Cu B ).…”

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confidence: 90%

Effective Pumping Proton Collection Facilitated by a Copper Site (CuB) of Bovine Heart Cytochrome c Oxidase, Revealed by a Newly Developed Time-resolved Infrared System

Kubo¹,

Nakashima²,

Yamaguchi

et al. 2013

Journal of Biological Chemistry

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Background: Cytochrome c oxidase reduces O 2 coupled with proton pumping. Results: A newly developed time-resolved infrared system reveals transient conformational changes in the proton-pumping pathway upon CO binding to Cu B in the O 2 reduction site. Conclusion: Cu B promotes proton collection and effective blockage of back-leak of pumping protons. Significance: These critical findings in bioenergetics stimulate the new infrared approach for mechanistic investigation of any other protein function.

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confidence: 90%

Effective Pumping Proton Collection Facilitated by a Copper Site (CuB) of Bovine Heart Cytochrome c Oxidase, Revealed by a Newly Developed Time-resolved Infrared System

Kubo¹,

Nakashima²,

Yamaguchi

et al. 2013

Journal of Biological Chemistry

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“…A further major challenge has arisen from structural studies of bovine mitochondrial CcO where a quite different redox-and ligand-sensitive "H" pathway (4, 10) has been suggested to provide the proton translocation path. Mutations in this H pathway in bovine CcO appear to uncouple proton translocation (10). An equivalent H pathway is present but not continuous in bacterial CcOs, but mutations in this region show no effect on coupling (11,12).…”

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confidence: 99%

Water molecule reorganization in cytochrome c oxidase revealed by FTIR spectroscopy

Maréchal

Rich

2011

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

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Although internal electron transfer and oxygen reduction chemistry in cytochrome c oxidase are fairly well understood, the associated groups and pathways that couple these processes to gated proton translocation across the membrane remain unclear. Several possible pathways have been identified from crystallographic structural models; these involve hydrophilic residues in combination with structured waters that might reorganize to form transient proton transfer pathways during the catalytic cycle. To date, however, comparisons of atomic structures of different oxidases in different redox or ligation states have not provided a consistent answer as to which pathways are operative or the details of their dynamic changes during catalysis. In order to provide an experimental means to address this issue, FTIR spectroscopy in the 3,560-3,800 cm −1 range has been used to detect weakly H-bonded water molecules in bovine cytochrome c oxidase that might change during catalysis. Full redox spectra exhibited at least four signals at 3,674(+), 3,638(+), 3,620(−), and 3,607(+) cm −1 . A more complex set of signals was observed in spectra of photolysis of the ferrous-CO compound, a reaction that mimics the catalytic oxygen binding step, and their D 2 O and H 2 18 O sensitivities confirmed that they arose from water molecule rearrangements. Fitting with Gaussian components indicated the involvement of up to eight waters in the photolysis transition. Similar signals were also observed in photolysis spectra of the ferrous-CO compound of bacterial CcO from Paracoccus denitrificans. Such water changes are discussed in relation to roles in hydrophilic channels and proton/electron coupling mechanism.energy coupling | mitochondria | respiratory chain | complex IV M itochondrial cytochrome c oxidase (CcO) catalyses electron transfer from cytochrome c to molecular oxygen, conserving the released energy as coupled transmembrane proton transfers. It is a member of a homologous "superfamily" of oxidases that includes diverse bacterial forms; all types in the CcO subgroup contain a common "catalytic core" of subunits I, II, and III with equivalent metal centers of Cu A , heme a. and a binuclear center (BNC) that is formed from heme a 3 and Cu B (1, 2). Structures of bovine mitochondrial (3, 4) and various bacterial (5-7) CcOs have been solved at atomic resolution and show common details in the binuclear heme a 3 ∕Cu B catalytic site, other cofactors, key amino acids, and several unusual structural features. These structural data, coupled with a substantial body of biophysical and spectroscopic studies, have provided detailed information on internal electron transfer and the chemical catalytic cycle of oxygen reduction.Crucially, however, these structural data have not provided a single picture for all forms of CcO of the pathway(s) for coupled proton transfer and, therefore, the interplay between electron transfer/oxygen chemistry and the intraprotein proton transfer reactions that result in proton-motive action. Crystal structures of bacteri...

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“…To this end, we have produced high-resolution structures of two defining mutants in the proton uptake routes identified as D and K pathways in RsCcO. Although these paths are apparently well conserved in most aa 3 -type oxidases, evidence from studies of bovine CcO has called into question whether they carry out the same function in all cases (7).…”

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Crystallographic and online spectral evidence for role of conformational change and conserved water in cytochrome oxidase proton pump

Liu

Qin

Ferguson‐Miller

2011

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

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Crystal structures in both oxidized and reduced forms are reported for two bacterial cytochrome c oxidase mutants that define the D and K proton paths, showing conformational change in response to reduction and the loss of strategic waters that can account for inhibition of proton transfer. In the oxidized state both mutants of the Rhodobacter sphaeroides enzyme, D132A and K362M, show overall structures similar to wild type, indicating no long-range effects of mutation. In the reduced state, the mutants show an altered conformation similar to that seen in reduced wild type, confirming this reproducible, reversible response to reduction. In the strongly inhibited D132A mutant, positions of residues and waters in the D pathway are unaffected except in the entry region close to the mutation, where a chloride ion replaces the missing carboxyl and a 2-Å shift in N207 results in loss of its associated water. In K362M, the methionine occupies the same position as the original lysine, but K362-and T359-associated waters in the wild-type structure are missing, likely accounting for the severe inhibition. Spectra of oxidized frozen crystals taken during X-ray radiation show metal center reduction, but indicate development of a strained configuration that only relaxes to a native form upon annealing. Resistance of the frozen crystal to structural change clarifies why the oxidized conformation is observable and supports the conclusion that the reduced conformation has functional significance. A mechanism is described that explains the conformational change and the incomplete response of the D-path mutant.conformational gating | membrane protein structure | proton path mutants | X-ray reduction | water chain C ytochrome c oxidase (CcO) is a major contributor to and regulator of energy conservation in eukaryotic and many prokaryotic organisms. It uses four electrons from cytochrome c and four protons from the inner side of the membrane to reduce O 2 to water (1). In each catalytic cycle, four protons are also translocated across the membrane to generate a membrane potential, but the mechanistic details of coupling between proton pumping and the electron transfer events are still not fully understood (1, 2). Our recently solved crystal structures of the fully reduced form of Rhodobacter sphaeroides (Rs) CcO reveal conformational changes that were not previously observed (3), introducing mechanistic possibilities for coupling and gating of proton transfer, the significance of which we further elucidate in this report.Bacterial forms of CcO are similar in many respects to the mammalian mitochondrial CcO and their simpler subunit composition and ease of mutation have made them useful model systems for studying the mechanism of energy conservation, assuming it to be conserved. It has become increasingly clear, however, that within the large superfamily of heme-copper oxidases some bacterial forms may have solved the proton pumping problem in different ways (4, 5), and even those most closely related to eukaryotic oxidases (the aa ...

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The proton pumping pathway of bovine heart cytochrome c oxidase

Cited by 133 publications

References 37 publications

Effective Pumping Proton Collection Facilitated by a Copper Site (CuB) of Bovine Heart Cytochrome c Oxidase, Revealed by a Newly Developed Time-resolved Infrared System

Effective Pumping Proton Collection Facilitated by a Copper Site (CuB) of Bovine Heart Cytochrome c Oxidase, Revealed by a Newly Developed Time-resolved Infrared System

Water molecule reorganization in cytochrome c oxidase revealed by FTIR spectroscopy

Crystallographic and online spectral evidence for role of conformational change and conserved water in cytochrome oxidase proton pump

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