Protonation Is Part of Dielectric Relaxation upon Reduction of Bacteriopheophytin in Reaction Center of Photosynthetic Bacterium Rhodobacter sphaeroides

The pH dependence of the free energy level of the flash induced primary charge pair P+IA– was determined by combination of the results from indirect charge recombination of P+QA– and from delayed fluorescence of the excited dimer (P*) in reaction center of photosynthetic bacterium Rhodobacter sphaeroides where the native ubiquinone at the primary quinone binding site QA was replaced by low potential anthraquinone derivatives. The following observations were made: 1) The free energy state of P+IA– was pH independent below pH 10 (–370 ± 10 meV relative to that of the excited dimer P*) and showed remarkable decrease (about 20 meV/pH unit) above pH 10. A not insignificant part of the dielectric relaxation of the P+IA– charge pair (about 120 meV) should come from protonation related changes. 2) The single exponential decay character of the kinetics proves that the protonated/unprotonated P+IA– and P+QA– states are in equilibria and the rate constants of protonation konH +koffH is much larger than that of the charge back reaction kback ~103 s-1. 3) Highly similar pH profiles were measured for the free energy states of P+QA– and P+IA–, indicating that the same acidic cluster around QB should response to both anionic species. This was supported by model calculations based on anticooperative proton distribution in the cluster with key residues of GluL212, AspL213, AspM17 and GluH173 and on effect of polarization of the aqueous phase on the electrostatic interaction. The larger distance of IA– from the cluster (25.2 Å) than that of QA‒ (14.5 Å) is compensated by smaller effective dielectric constant (6.5 ± 0.5 and 10.0 ± 0.5, respectively). 4) The P* → P+QA− and IA‒QA → IAQA− electron transfers are enthalpy driven reactions with exemption of very large (>60%) or negligible entropic contributions in cases of substitution by 2,3-dimethyl-AQ or 1-chloro-AQ, respectively. The possible structural consequences are discussed.