Identification of the proton pathway in bacterial reaction centers:: Replacement of Asp-M17 and Asp-L210 with Asn reduces the proton transfer rate in the presence of Cd2+

The reaction center (RC) from Rhodobacter sphaeroides converts light into chemical energy through the reduction and protonation of a bound quinone molecule Q(B) (the secondary quinone electron acceptor). We investigated the proton transfer pathway by measuring the proton-coupled electron transfer, k(AB)((2)) [Q(A)(radical anion)Q(B)(radical anion) + H+ --> Q(A)(Q(B)H)(-)] in native and mutant RCs in the absence and presence of Cd2+. Previous work has shown that the binding of Cd2+ decreases k(AB)((2)) in native RCs approximate to 100-fold, The preceding paper shows that bound Cd2+ binds to Asp-H124 His-H126, and His-H128, This region represents the entry point for protons. In this work we investigated the proton transfer pathway connecting the entry point with Q(B)(radical anion) by searching for mutations that greatly affect k(AB)((2)) (greater than or similar to 10-fold) in the presence of Cd2+, where k(AB)((2)) is limited by the proton transfer rate (k(H)) Upon mutation of Asp-L210 or Asp-M17 to Asn, k(H) decreased from approximate to 60 s(-1) to approximate to 7 s(-1), which shows the important role that Asp-L210 and Asp-M17 play in the proton transfer chain. By comparing the rate of proton transfer in the mutants (k(H) approximate to 7 s(-1)) With that in native RCs in the absence of Cd2+ (k(H) greater than or equal to 10(4) s(-1)), we conclude that alternate proton transfer pathways, which have been postulated, are at least 10(3)-fold less effective.