Effect of pH and surface potential on the rate of electric potential generation due to proton uptake by secondary quinone acceptor of reaction centers in Rhodobacter sphaeroides chromatophores

An electrometric method was used to investigate the effect of pH and ionic strength on the second flash-induced formation of the transmembrane electric potential difference (A~b) arising from proton uptake by the quinone complex of the photosynthetic reaction center (RC) in chromatophores of non-sulphur purple bacterium Rhodobacter sphaeroides (wild-type). The characteristic time of this A~b phase is approx. 70 p.s at pH 7.5 in the presence of 20 mM KC1. Increases in both pH and salt concentration were found to slow the time of generation of the electric potential due to quinol formation (QBH2) after the second flash by more than 10-times. At higher pH, the effects of salts were more pronounced. The pH dependence of the Q~protonation rate is explained by the change in the surface proton concentration near the RC protein. The density of surface charges calculated on the basis of Gouy-Chapman theory is approx. 0.002 e/.~ 2 (pH 7) or approx. 10 negative charges per RC protein from the cytoplasmic side of the membrane, in good agreement with previous estimates for chromatophores. The sensitivity of the rate constant of the second flash-induced RC protonation to the salt concentration probably resolves the contradiction among results reported by different laboratories (Wraight, C.A. (1979) Biochim. Biophys. Acta 548, 309-327; Kleinfeld, D., Okamura, M.Y. and Feher, G. (1985) Biochim. Biophys. Acta 809, 291-310) for the pH dependence of the rate constant of the reaction QAQa ~ QAQBH2 •