Intermediates in the Reaction of Reduced Cytochrome Oxidase with Dioxygena

Orii, Yutaka

doi:10.1111/j.1749-6632.1988.tb35327.x

Cited by 72 publications

(74 citation statements)

References 40 publications

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“…53. Briefly, the wild-type R. sphaeroides CytcO at a concentration of 10 M in 100 mM Hepes at pH 7.4 and 0.1% n-dodecyl-␤-D-maltoside was reduced with a slight excess of dithionite and transferred anaerobically to one of the syringes of the stopped flow apparatus.…”

Section: Methodsmentioning

confidence: 99%

Plasticity of Proton Pathway Structure and Water Coordination in Cytochrome c Oxidase

Namslauer

Lepp

Brändén

et al. 2007

Journal of Biological Chemistry

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Cytochrome c oxidase (CytcO) is a redox-driven, membranebound proton pump. One of the proton transfer pathways of the enzyme, the D pathway, used for the transfer of both substrate and pumped protons, accommodates a network of hydrogenbonded water molecules that span the distance between an aspartate (Asp 132 ), near the protein surface, and glutamate Glu 286 , which is an internal proton donor to the catalytic site. To investigate how changes in the environment around Glu 286 affect the mechanism of proton transfer through the pathway, we introduced a non-hydrogen-bonding (Ala) or an acidic residue (Asp) at position Ser 197 (S197A or S197D), located ϳ7 Å from Glu 286 . Although Ser 197 is hydrogen-bonded to a water molecule that is part of the D pathway "proton wire," replacement of the Ser by an Ala did not affect the proton transfer rate. In contrast, the S197D mutant CytcO displayed a turnover activity of ϳ35% of that of the wild-type CytcO, and the O 2 reduction reaction was not linked to proton pumping. Instead, a fraction of the substrate protons was taken from the positive ("incorrect") side of the membrane. Furthermore, the pH dependence of the proton transfer rate was altered in the mutant CytcO. The results indicate that there is plasticity in the water coordination of the proton pathway, but alteration of the electrostatic potential within the pathway results in uncoupling of the proton translocation machinery.

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Section: Methodsmentioning

confidence: 99%

Plasticity of Proton Pathway Structure and Water Coordination in Cytochrome c Oxidase

Namslauer

Lepp

Brändén

et al. 2007

Journal of Biological Chemistry

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“…When the production of P is carried out in the presence of 18 O 2 we find that one equivalent of H 2 18 O appears in the solvent. This observation implies that the O-O bond is split and that one of the oxygen atoms thus produced is either released as H 2 O or can equilibrate with H 2 O in the solvent.…”

mentioning

confidence: 94%

“…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.…”

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confidence: 99%

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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.

123

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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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“…In this case, the 'energy' term was represented by the high electrochemical proton gradient µ [54,55]. Furthermore, the existence of the ferryl compound structure was proved using Raman spectroscopy in which the iron-oxygen vibration was observed at 790 cm −1 [56][57][58].…”

Section: Assessment Of Cox Transitional Statesmentioning

confidence: 99%

Fabrication of biomolecule–copolymer hybrid nanovesicles as energy conversion systems

Ho¹,

Chu²,

Lee³

et al. 2005

Nanotechnology

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This work demonstrates the integration of the energy-transducing proteins bacteriorhodopsin (BR) from Halobacterium halobium and cytochrome c oxidase (COX) from Rhodobacter sphaeroides into block copolymeric vesicles towards the demonstration of coupled protein functionality. An ABA triblock copolymer-based biomimetic membrane possessing UV-curable acrylate endgroups was synthesized to serve as a robust matrix for protein reconstitution. BR-functionalized polymers were shown to generate light-driven transmembrane pH gradients while pH gradient-induced electron release was observed from COX-functionalized polymers. Cooperative behaviour observed from composite membrane functionalized by both proteins revealed the generation of microamp-range currents with no applied voltage. As such, it has been shown that the fruition of technologies based upon bio-functionalizing abiotic materials may contribute to the realization of high power density devices inspired by nature.

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Intermediates in the Reaction of Reduced Cytochrome Oxidase with Dioxygena

Cited by 72 publications

References 40 publications

Plasticity of Proton Pathway Structure and Water Coordination in Cytochrome c Oxidase

Plasticity of Proton Pathway Structure and Water Coordination in Cytochrome c Oxidase

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

Fabrication of biomolecule–copolymer hybrid nanovesicles as energy conversion systems

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