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 Cd21

The reaction center (RC) from Rhodobacter sphaeroides converts light into chemical energy through the reduction and protonation of a bound quinone molecule QB (the secondary quinone electron acceptor). We investigated the proton transfer pathway by measuring the proton-coupled electron transfer, kAB [QA.QB. 1 H13 QA(QBH)] in native and mutant RCs in the absence and presence of Cd21. Previous work has shown that the binding of Cd21 decreases kAB in native RCs '100-fold. The preceding paper shows that bound Cd21 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 QB. by searching for mutations that greatly affect kAB (*10-fold) in the presence of Cd21, where kAB is limited by the proton transfer rate (kH). Upon mutation of Asp-L210 or Asp-M17 to Asn, kH decreased from '60 s21 to '7 s21, 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 (kH ' 7 s21) with that in native RCs in the absence of Cd21 (kH > 104 s21), we conclude that alternate proton transfer pathways, which have been postulated, are at least 103-fold less effective.