GLUTAMATE-286 IN CYTOCHROME AA(3) FROM RHODOBACTER-SPHAEROIDES IS INVOLVED IN PROTON UPTAKE DURING THE REACTION OF THE FULLY-REDUCED ENZYMEWITH DIOXYGEN

The reaction with dioxygen of solubilized fully-reduced wild-type and EQ(I-286) (exchange of glutamate 286 of subunit I for glutamine) mutan t cytochrome c oxidase from Rhodobacter sphaeroides has been studied u sing the flow-flash technique in combination with optical absorption s pectroscopy. Proton uptake was measured using a pH-indicator dye. In a ddition, internal electron-transfer reactions were studied in the abse nce 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 ox ygen-binding binuclear center that is clearly a part of the pathway. T he 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 fo rmation of the peroxy intermediate is impaired, Cu-A 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 p eroxy intermediate and transfer of the fourth electron to the binuclea r center. The results also indicate that the proton uptake associated with formation of the ferryl intermediate controls the electron transf er from Cu-A to heme a.