Effects of mutation of the conserved glutamic acid-286 in subunit I of cytochrome c oxidase from Rhodobacter sphaeroides

We have studied the effects of mutations, E286Q and E286D, of the conserved glutamate in subunit I of cytochrome c oxidase from Rhodobacter sphaeroide s with a view to evaluating the role of this residue in redox-linked proton translocation, The mutation E286D did not have any dramatic effects on enz yme properties and retained 50% of wild-type catalytic activity. For E286Q a fi action of the binuclear center was trapped in an unreactive, spectrall y distinct form which is most likely due to misfolded protein, but the majo rity of E286Q reacted normally with formate and cyanide in the oxidized sta te, and with carbon monoxide and cyanide in the dithionite-reduced form. Th e mutation also had little effect on the pH-dependent redox properties of h aem a in the reactive fraction. However, formation of the P state from oxid ized enzyme with hydrogen peroxide or by aerobic incubation with carbon mon oxide was inhibited. In particular, only an F-type product was obtained, at less than 25% yield, in the reaction with hydrogen peroxide. The aerobic s teady state in the presence of ferrous cytochrome c was characterized by es sentially fully reduced haem a and ferric haem a(3), suggesting that the mu tation hinders electron transfer from haem a to the binuclear center. Under these conditions or after reoxidation, on a seconds time scale, of haem a( 3) following anaerobiosis, there was no indication of accumulation of signi ficant amounts of P state. We propose that the glutamate is implicated in s everal steps in the catalytic cycle, O --> R, P --> F, and, possibly, F --> O. The results are discussed in relation to the "glutamate trap" model for proton translocation.