EVIDENCE THAT SERINE-L223 IS INVOLVED IN THE PROTON-TRANSFER PATHWAY TO Q(B) IN THE PHOTOSYNTHETIC REACTION CENTER OF RHODOPSEUDOMONAS-VIRIDIS

In the reaction center of purple photosynthetic bacteria, the reducing equivalents produced by primary charge separation are exported via an ubiquinone molecule working as a two-electron shuttle. This loosely-bound quinone, called Q(B), accepts in successive flashes two electrons from the tightly bound primary quinone acceptor Q(A), along with two protons from the external medium. The surrounding protein plays an important role in stabilizing the semiquinone anion and in providing a pathway for protons from the cytoplasmic phase to Q(B). Herbicides of the triazine type compete with Q(B) for the binding pocket and their binding is controlled by nearby amino acid residues. We have studied the kinetics of the first and second electron transfer from Q(A) to Q(B) in two herbicide-resistant mutants from Rhodopseudomonas viridis, T1 (Arg L217 --> His, Ser L223 --> Ala) and MAV5 (Arg L217 --> His, Val L220 --> Leu), in order to determine whether these residues are involved in proton transfer to the reduced Q(B). The main effect of the mutant T1 was a drastic (600-fold at pH 7) decrease in the rate of the second electron transfer to Q(B) compared to the wild type. In contrast, the rate of the second electron transfer in the mutant MAV5 was decreased only slightly (10-fold) in the pH range from 7 to 11. We attribute the inhibition of the second electron transfer in the Ser L223 --> Ala mutation to an essential role of Ser L223 in the donation of the first proton to the reduced Q(B). This further corroborates the thesis that the transfer of the second electron is intimately related to the transfer of the first proton to Q(B) and that any inhibition of proton supply will be transformed into an inhibition of the transfer of the second electron. The pH dependence of the electron transfer kinetics may indicate that the second mutated residue in the mutant T1, His L217, plays a role as auxiliary proton donor. We suggest that a cluster of hydrogen bonds involving Ser L223 and the quinone carbonyl oxygen could be important for efficient protonation of Q(B). This cluster is lost in the absence of Ser L223.