Electron and proton transfer on the acceptor side of the reaction center in chromatophores of Rhodobacter capsulatus:: Evidence for direct protonation of the semiquinone state of QB

1. The absorption changes associated with the formation of P(+)Q(B)(red) (Q(B)(red) stands for the semiquinone state of the secondary quinone acceptor) were investigated in chromatophores of Rhodobacter capsulatus. Marked modifications of the semiquinone spectrum were observed when the pH was lowered from 7 to 5. These modifications match those expected for a complete conversion of Q(B)(red) from the anionic state Q(B)(-) at pH 7 to the neutral protonated state Q(B)H at pH 5. Similar modifications were observed in chromatophores from Rb. sphaeroides, but not in purified reaction centers from Rb. capsulatus, suggesting that the environment of the reaction center (native membrane vs detergent micelle) is the crucial parameter. 2. The recombination reaction P(+)Q(B)(red -->) PQ(B) was investigated as a function of pH. No particular kinetic heterogeneity was observed at low pH, showing that Q(B)H remains mostly bound to the reaction center. The rate constant reaches a minimum value of 0.08 s(-1) at pH 6, suggesting that the direct route for recombination prevails in chromatophores below this pH, instead of the usual pathway via Q(A)(-). 3. The proton uptake caused by Q(B)(red) is about 1 below pH 7 and decreases at higher pH. It is suggested that the pH dependence of the conversion of Q(B)(-) to Q(B)H, occurring in a range where the uptake is constant, cannot be accommodated by a purely electrostatic model, but probably involves a conformational change. 4. The kinetics of the electron-transfer reaction Q(A)(-)Q(B)-->Q(A)Q(B)(red) were investigated. A 2-fold acceleration was observed between pH 7 and pH 5 (t(1/2) approximate to 30 and 15 mu s, respectively). A fast (<<10 mu s) unresolved phase appears to be present at both pHs. The second electron-transfer Q(A)(-)Q(B)(red-->)Q(A)Q(B)H(2) proceeds with a similar rate as the first electron transfer (15-30 mu s phase). Consequences for the rate-limiting step are discussed. 5. The carotenoid shift, indicative of the membrane potential, displays a rising phase concomitant with the Q(A)(-)Q(B)(-->)Q(A)Q(B)(red) electron transfer. Its relative extentis markedly increased at pH 5, with part of the kinetics occurring during the unresolved fast phase. 6. The extent of the electrochromic shift of bacteriopheophytin around 750 nm associated with formation of Q(B)(red) decreases toward acidic pH, reflecting the charge compensation due to proton uptake and the formation of neutral Q(B)H.