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 Cd(2+)

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)⨪Q(B)⨪ + H(+) → Q(A)(Q(B)H)(−)] in native and mutant RCs in the absence and presence of Cd(2+). Previous work has shown that the binding of Cd(2+) decreases k(AB)((2)) in native RCs ≈100-fold. The preceding paper shows that bound Cd(2+) 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)⨪ by searching for mutations that greatly affect k(AB)((2)) (≳10-fold) in the presence of Cd(2+), 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 ≈60 s(−1) 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) ≈ 7 s(−1)) with that in native RCs in the absence of Cd(2+) (k(H) ≥ 10(4) s(−1)), we conclude that alternate proton transfer pathways, which have been postulated, are at least 10(3)-fold less effective.