A CONSERVED GLUTAMIC-ACID IN HELIX-VI OF CYTOCHROME BO(3) INFLUENCES A KEY STEP IN OXYGEN REDUCTION

We have compared the reactions with dioxygen of wild-type cytochrome b o(3) and a mutant in which a conserved glutamic acid at position-286 o f subunit I has been changed to an alanine. Flow-flash experiments rev eal that oxygen binding and the rate of heme-heme electron transfer ar e unaffected by the mutation. Reaction of the fully (3-electron) reduc ed mutant cytochrome bos with dioxygen yields a binuclear center which is substantially in the P (peroxy) state, not the well-characterized F (oxyferryl) state which is the product of the reaction of the fully reduced wild-type enzyme with dioxygen [Puustinen, A., et al. (1996) P roc. Natl. Acad. Sci. U.S.A. 93, 1545-1548]. These results confirm tha t proton uptake is important in controlling the later stages of dioxyg en reduction in heme-copper oxidases and show that E286 is an importan t component of the channel that delivers these protons to the active s ite.