Reduction and protonation of the secondary quinone acceptor of Rhodobactersphaeroides photosynthetic reaction center: kinetic model based on a comparison of wild-type chromatophores with mutants carrying Arg -> Ile substitution at sites 207 and 217 in the L-subunit

After the light-induced charge separation in the photosynthetic reaction ce nter (RC) of Rhodobacter sphaeroides, the electron reaches, via the tightly bound ubiquinone Q(A), the loosely bound ubiquinone Q(B). After two subseq uent flashes of light, Q(B) is reduced to ubiquinol Q(B)H(2). With a semiqu inone anion Q(B)(-) formed as an intermediate after the first flash. We stu died Q(B)H(2) formation in chromatophores from Rb. sphaeroides mutants that carried Arg --> Ile substitution at sites 207 and 217 in the L-subunit. Wh ile Arg-L207 is 17 Angstrom away from Q(B), Arg-L217 is closer (9 Angstrom) and contacts the Q(B)-binding pocket. From the pH dependence of the charge recombination in the RC after the first flash, we estimated Delta G(AB), t he free energy difference between the Q(A)(-)Q(B) and Q(A)Q(B)(-) states, a nd pK(212), the apparent pK of Glu-L212, a residue that is only 4 Angstrom away from Q(B). As expected, the replacement of positively charged arginine s by neutral isoleucines destabilized the Q(B)(-) state in the L217RI mutan t to a larger extent than in the L207RI one. Also as expected, pK(212) incr eased by similar to 0.4 pH units in the L207RI mutant. The value of pK(212) in the L217RI mutant decreased by 0.3 pH units, contrary to expectations. The rate of the Q(A)(-)Q(B)(-) --> Q(A)Q(B)H(2) transition upon the second flash, as monitored by electrometry via the accompanying changes in the mem brane potential, was two times faster in the L207RI mutant than in the wild -type, but remained essentially unchanged in the L217RI mutant. To rational ize these findings, we developed and analyzed a kinetic model of the Q(A)(- )Q(B)(-) --> Q(A)Q(B)H(2) transition. The model properly described the avai lable experimental data and provided a set of quantitative kinetic and ther modynamic parameters of the Q(B) turnover. The non-electrostatic, 'chemical ' affinity of the Q(B) Site to protons proved to be as important for the at tracting protons from the bulk, as the appropriate electrostatic potential. The mutation-caused changes in the chemical proton affinity could be estim ated from the difference between the experimentally established pK(212) shi fts and the expected changes in the electrostatic potential at Glu-L212, ca lculable from the X-ray structure of the RC. Based on functional studies, s tructural data and kinetic modeling, we suggest a mechanistic scheme of the Q(B) turnover. The detachment of the formed ubiquinol from its proximal po sition next to Glu-L212 is considered as the rate-limiting step of the reac tion cycle. (C) 2000 Elsevier Science B.V. All rights reserved.