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.
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.
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