Electron and proton transfer on the acceptor side of the reaction center in chromatophores of Rhodobacter capsulatus: Evidence for direct protonationof the semiquinone state of Q(B)

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 mod ifications of the semiquinone spectrum were observed when the pH was lowere d from 7 to 5. These modifications match those expected for a complete conv ersion of Q(B)(red) from the anionic state Q(B)(-) at pH 7 to the neutral p rotonated state Q(B)H at pH 5. Similar modifications were observed in chrom atophores from Rb. sphaeroides, but not in purified reaction centers from R b. capsulatus, suggesting that the environment of the reaction center (nati ve membrane vs detergent micelle) is the crucial parameter. 2. The recombin ation 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 reache s a minimum value of 0.08 s(-1) at pH 6, suggesting that the direct route f or recombination prevails in chromatophores below this pH, instead of the u sual 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 depen dence 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 ele ctron-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 ri sing phase concomitant with the Q(A)(-)Q(B)(-->)Q(A)Q(B)(red) electron tran sfer. Its relative extentis markedly increased at pH 5, with part of the ki netics occurring during the unresolved fast phase. 6. The extent of the ele ctrochromic shift of bacteriopheophytin around 750 nm associated with forma tion of Q(B)(red) decreases toward acidic pH, reflecting the charge compens ation due to proton uptake and the formation of neutral Q(B)H.