Glutamate 286 in cytochrome aa3 from Rhodobacter sphaeroides is involved in proton uptake during the reaction of the fully-reduced enzyme with dioxygen.
The reaction with dioxygen of solubilized fully-reduced wild-type and EQ(I-286) (exchange of glutamate 286 of subunit I for glutamine) mutant cytochrome c oxidase from Rhodobacter sphaeroides has been studied using the flow-flash technique in combination with optical absorption spectroscopy. Proton uptake was measured using a pH-indicator dye. In addition, internal electron-transfer reactions were studied in the absence of oxygen. Glutamate 286 is found in a proton pathway proposed to be used for pumped protons from the crystal structure of cytochrome c oxidase from Paracoccus denitrificans [Iwata et al. (1995) Nature 376, 660-669; E278 in P.d. numbering]. It is the residue closest to the oxygen-binding binuclear center that is clearly a part of the pathway. The results show that the wild-type enzyme becomes fully oxidized in a few milliseconds at pH 7.4 and displays a biphasic proton uptake from the medium. In the EQ(I-286) mutant enzyme, electron transfer after formation of the peroxy intermediate is impaired, CuA remains reduced, and no protons are taken up from the medium. Thus, the results suggest that E(I-286) is necessary for proton uptake after formation of the peroxy intermediate and transfer of the fourth electron to the binuclear center. The results also indicate that the proton uptake associated with formation of the ferryl intermediate controls the electron transfer from CuA to heme a.
Adelroth
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Department of Biochemistry and Biophysics, Göteborg University, Sweden.
Ek
M S
MS
Mitchell
D M
DM
Gennis
R B
RB
Brzezinski
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D019943:Amino Acid Substitution / Q000235:genetics; D002248:Carbon Monoxide / Q000378:metabolism; D002384:Catalysis; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism*; D005971:Glutamates / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D011522:Protons; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics
D005971:Glutamates; D011522:Protons; D002248:Carbon Monoxide; D003576:Electron Transport Complex IV; D010100:Oxygen