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.
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.
Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, United States.
Istvan
I
Szundi
Chie
C
Funatogawa
Jennifer
J
Cassano
William
W
McDonald
Jayashree
J
Ray
Carrie
C
Hiser
Shelagh
S
Ferguson-Miller
Robert B
RB
Gennis
Ólöf
Ó
Einarsdóttir
D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010100:Oxygen / Q000378:metabolism*; D012242:Rhodobacter sphaeroides / Q000201:enzymology*; D013053:Spectrophotometry / Q000379:methods*
D003576:Electron Transport Complex IV; D010100:Oxygen