PATHWAY OF PROTON-TRANSFER IN BACTERIAL REACTION CENTERS - REPLACEMENT OF SERINE-L223 BY ALANINE INHIBITS ELECTRON AND PROTON TRANSFERS ASSOCIATED WITH REDUCTION OF QUINONE TO DIHYDROQUINONE

The pathway of proton transfer in the reaction center (RC) from Rhodobacter sphaeroides was investigated by site-directed mutagenesis. Ser-L223, a putative proton donor that forms a hydrogen bond with the secondary quinone acceptor Q(B), was replaced with Ala and Thr. RCs with Ala-L223 displayed reduced electron transfer and proton uptake rates in the reaction Q-/(A)Q-/(B) + 2H+ → Q(A)Q(B)H2. The rate constant for this reaction, k(2)/(AB), was found to be reduced ≃350-fold to 4.0 ± 0.2 s-1. Proton uptake measurements using a pH indicator dye showed a rapid uptake of 1 H+ per RC followed by a slower uptake of 1 H+ per RC at a rate of 4.1 ± 0.1s-1; native RCs showed a rapid uptake of 2H+ per RC. Evidence is provided that these changes were not due to gross structural changes in the binding site of Q(B). RCs with Thr-L223 showed little reduction in the rates of electron and proton transfer. These results indicate that proton transfer from the hydroxyl group of Ser-L223 or Thr-L223 is required for fast electron and proton transfer associated with the formation of the dihydroquinone QH2. In contrast, previous work showed that replacing Glu-L212, another putative proton donor to Q(B), with Gln slowed proton uptake from solution without significantly altering electron transfer. We propose a model that involves two distinct proton transfer steps. The first step occurs prior to transfer of the second electron to Q(B) and involves proton transfer from Ser-L223. The second step occurs after this electron transfer through a pathway involving Glu-L212.