Reduction and protonation of the secondary quinone acceptor of Rhodobacter sphaeroides 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

Abstract

After the light-induced charge separation in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides, the electron reaches, via the tightly bound ubiquinone Q A, the loosely bound ubiquinone Q B. After two subsequent flashes of light, Q B is reduced to ubiquinol Q BH 2, with a semiquinone anion Q B − formed as an intermediate after the first flash. We studied Q BH 2 formation in chromatophores from Rb. sphaeroides mutants that carried Arg→Ile substitution at sites 207 and 217 in the L-subunit. While Arg-L207 is 17 Å away from Q B, Arg-L217 is closer (9 Å) and contacts the Q B-binding pocket. From the pH dependence of the charge recombination in the RC after the first flash, we estimated Δ G AB, the free energy difference between the Q A −Q B and Q AQ B − states, and p K 212, the apparent p K of Glu-L212, a residue that is only 4 Å away from Q B. As expected, the replacement of positively charged arginines by neutral isoleucines destabilized the Q B − state in the L217RI mutant to a larger extent than in the L207RI one. Also as expected, p K 212 increased by ∼0.4 pH units in the L207RI mutant. The value of p K 212 in the L217RI mutant decreased by 0.3 pH units, contrary to expectations. The rate of the Q A −Q B −→Q AQ BH 2 transition upon the second flash, as monitored by electrometry via the accompanying changes in the membrane potential, was two times faster in the L207RI mutant than in the wild-type, but remained essentially unchanged in the L217RI mutant. To rationalize these findings, we developed and analyzed a kinetic model of the Q A −Q B −→Q AQ BH 2 transition. The model properly described the available experimental data and provided a set of quantitative kinetic and thermodynamic 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 attracting protons from the bulk, as the appropriate electrostatic potential. The mutation-caused changes in the chemical proton affinity could be estimated from the difference between the experimentally established p K 212 shifts and the expected changes in the electrostatic potential at Glu-L212, calculable from the X-ray structure of the RC. Based on functional studies, structural data and kinetic modeling, we suggest a mechanistic scheme of the Q B turnover. The detachment of the formed ubiquinol from its proximal position next to Glu-L212 is considered as the rate-limiting step of the reaction cycle.