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Spectral identification of intermediates generated during the reaction of dioxygen with the wild-type and EQ(I-286) mutant of Rhodobacter sphaeroides cytochrome c oxidase.
Szundi, Istvan; Funatogawa, Chie; Cassano, Jennifer; McDonald, William; Ray, Jayashree; Hiser, Carrie; Ferguson-Miller, Shelagh; Gennis, Robert B; Einarsdóttir, Ólöf.
Affiliation
  • Szundi I; Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, United States.
Biochemistry ; 51(46): 9302-11, 2012 Nov 20.
Article in En | MEDLINE | ID: mdl-23057757
Cytochrome c oxidase from Rhodobacter sphaeroides is frequently used to model the more complex mitochondrial enzyme. The O(2) reduction in both enzymes is generally described by a unidirectional mechanism involving the sequential formation of the ferrous-oxy complex (compound A), the P(R) state, the oxyferryl F form, and the oxidized state. In this study we investigated the reaction of dioxygen with the wild-type reduced R. sphaeroides cytochrome oxidase and the EQ(I-286) mutant using the CO flow-flash technique. Singular value decomposition and multiexponential fitting of the time-resolved optical absorption difference spectra showed that three apparent lifetimes, 18 µs, 53 µs, and 1.3 ms, are sufficient to fit the kinetics of the O(2) reaction of the wild-type enzyme. A comparison of the experimental intermediate spectra with the corresponding intermediate spectra of the bovine enzyme revealed that P(R) is not present in the reaction mechanism of the wild-type R. sphaeroides aa(3). Transient absorbance changes at 440 and 610 nm support this conclusion. For the EQ(I-286) mutant, in which a key glutamic residue in the D proton pathway is replaced by glutamine, two lifetimes, 16 and 108 µs, were observed. A spectral analysis of the intermediates shows that the O(2) reaction in the EQ(I-286) mutant terminates at the P(R) state, with 70% of heme a becoming oxidized. These results indicate significant differences in the kinetics of O(2) reduction between the bovine and wild-type R. sphaeroides aa(3) oxidases, which may arise from differences in the relative rates of internal electron and proton movements in the two enzymes.
Subject(s)

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Oxygen / Spectrophotometry / Rhodobacter sphaeroides / Electron Transport Complex IV / Mutation Type of study: Diagnostic_studies Language: En Journal: Biochemistry Year: 2012 Document type: Article Affiliation country: United States Country of publication: United States

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Oxygen / Spectrophotometry / Rhodobacter sphaeroides / Electron Transport Complex IV / Mutation Type of study: Diagnostic_studies Language: En Journal: Biochemistry Year: 2012 Document type: Article Affiliation country: United States Country of publication: United States