The involvement of the fully oxidized state in cytochrome oxidase reaction with oxygen studied with the 655 nm band as a probe

Energization of isolated rat liver mitochondria with ATP under conditions in which cytochrome c is poised in a highly oxidized state shifts the state of cytochrome oxidase (cytochrome c oxidase; ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) from fully oxidized to two new spectroscopically distinguishable states depending on the applied phosphorylation potential and redox potential at cytochrome c. Both new states are spectrally similar or identical to two previously described intermediates in the reaction between reduced enzyme and O2. The data suggest that the energy-dependent transitions are due to reversed electron transfer from water to ferricytochrome c linked to accumulation of intermediates of O2 reduction at the catalytic heme a3/copper center. Titrations with redox potential indicate that each transition is a one-electron step, a finding that would identify the second observed compound as enzyme-bound peroxide or its equivalent. This is consistent with this compound being spectrally identical to "Compound C," previously described as the reaction product between half-reduced oxidase (two electrons) and O2. On the basis of these data a catalytic scheme of O2 reduction is proposed for the heme a3/copper center of cytochrome oxidase.

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“…3) is inconsistent with the idea that it contains ferrous heme a3 (8,10,11). The lack ofa sizable Soret band (see ref.…”

Section: Resultsmentioning

confidence: 97%

Energy-dependent reversal of the cytochrome oxidase reaction.

Wikström¹

1981

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

149

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“…Vol. 217 +Na2S204 5nm shows a decrease in absorption around 650-660nm, indicating an absence of the 655nm absorption band usually present in the unliganded ferric enzyme (Denis, 1977(Denis, , 1981Chance & Leigh, 1977;Clore et al, 1980). A possibility that is consistent with these findings is that the direct reaction of H202 with the oxidized enzyme generates a 'peroxy' complex in accordance with eqn.…”

Section: Discussionmentioning

confidence: 99%

Formation and reduction of a ‘peroxy’ intermediate of cytochrome c oxidase by hydrogen peroxide

Wrigglesworth¹

1984

Biochemical Journal

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In the presence of micromolar concentrations of H2O2, ferric cytochrome c oxidase forms a stable complex characterized by an increased absorption intensity at 606-607 nm with a weaker absorption band in the 560-580 nm region. Higher (millimolar) concentrations of H2O2 result in an enzyme exhibiting a Soret band at 427 nm and an alpha-band of increased intensity in the 589-610 nm region. Addition of H2O2 to ferric cytochrome c oxidase in the presence of cyanide results in absorbance increases at 444nm and 605nm. These changes are not seen if H2O2 is added to the cyanide complex of the ferric enzyme. The results support the idea that direct reaction of H2O2 with ferric cytochrome a 3 produces a 'peroxy' intermediate that is susceptible to further reduction by H2O2 at higher peroxide concentrations. Electron flow through cytochrome a is not involved, and the final product of the reaction is the so-called 'pulsed' or 'oxygenated' ferric form of the enzyme.

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“…Later Greenwood et al suggested that the oxidation product of the mixed valence state is similar to oxygenated oxidase (105). Using Chance's triple trapping method, Denis (148) has shown that the 655-nm band is present when starting from the fu lly reduced state but when starting with the mixed valence state , the 655-nm band is abolished. These results together with our finding that the 655-nm band is abolished by the addition of H202 to the pulsed oxidase shows that the 428-nm form is produced when the experiment is initiated from the mixed valence state.…”

Section: Fully Reoxidized States Of Cytochrome Oxidasementioning

confidence: 99%