Document Detail


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.
MedLine Citation:
PMID:  23057757     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Istvan Szundi; Chie Funatogawa; Jennifer Cassano; William McDonald; Jayashree Ray; Carrie Hiser; Shelagh Ferguson-Miller; Robert B Gennis; Ólöf Einarsdóttir
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2012-11-06
Journal Detail:
Title:  Biochemistry     Volume:  51     ISSN:  1520-4995     ISO Abbreviation:  Biochemistry     Publication Date:  2012 Nov 
Date Detail:
Created Date:  2012-11-20     Completed Date:  2013-01-22     Revised Date:  2014-03-19    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  9302-11     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Electron Transport Complex IV / genetics,  metabolism*
Mutation*
Oxygen / metabolism*
Rhodobacter sphaeroides / enzymology*
Spectrophotometry / methods*
Grant Support
ID/Acronym/Agency:
GM26916/GM/NIGMS NIH HHS; GM53788/GM/NIGMS NIH HHS; HL16101/HL/NHLBI NIH HHS; R01 GM026916/GM/NIGMS NIH HHS; R01 GM053788/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
EC 1.9.3.1/Electron Transport Complex IV; S88TT14065/Oxygen
Comments/Corrections

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