Proton transfer in cytochrome bd-I from E. coli involves Asp-105 in CydB.

Cytochrome bds are bacterial terminal oxidases expressed under low oxygen conditions, and they are important for the survival of many pathogens and hence potential drug targets. The largest subunit CydA contains the three redox-active cofactors heme b, heme b and the active site heme d. One suggested proton transfer pathway is found at the interface between the CydA and the other major subunit CydB. Here we have studied the O reduction mechanism in E. coli cyt. bd-I using the flow-flash technique and focused on the mechanism, kinetics and pathway for proton transfer. Our results show that the peroxy (P) to ferryl (F) transition, coupled to the oxidation of the low-spin heme b is pH dependent, with a maximum rate constant (~10 s) that is slowed down at higher pH. We assign this behavior to rate-limitation by internal proton transfer from a titratable residue with pK ~ 9.7. Proton uptake from solution occurs with the same P➔F rate constant. Site-directed mutagenesis shows significant effects on catalytic turnover in the CydB variants Asp58➔Asn and Asp105➔Asn variants consistent with them playing a role in proton transfer. Furthermore, in the Asp105➔Asn variant, the reactions up to P formation occur essentially as in the wildtype bd-I, but the P➔F transition is specifically inhibited, supporting a direct and specific role for Asp105 in the functional proton transfer pathway in bd-I. We further discuss the possible identity of the high pK proton donor, and the conservation pattern of the Asp-105 in the cyt. bd superfamily.