Abstract
The proton uptake associated with the light-induced transfer of an electron to the acceptor quinones Q A and Q B was investigated in reaction centers from Rhodobacter sphaeroides R-26. The proton uptake was found to be pH dependent with maximum values of approx. 0.5 H +/e − at pH 9 for DQ A − and approx. 0.8 H +/e − at pH 10 for DQ AQ B −. The quinones are not protonated directly. The observed proton uptake is due to shifts in the p K values of amino acid residues that interact with the quinones. The pH-dependences of the proton uptake were fitted with a phenomenological model in which the protons are taken up by four amino acid residues. The deduced p K shifts associated with the reductions of the quinones ranged from 0.4 to 0.8 for Q A and from 0.4 to 1.3 for Q B. The proton uptake by D +Q A − and D +Q AQ B − was less than that by DQ A − and DQ AQ B −, respectively, indicating a release of protons associated with the formation of D +. To calculate the pH-dependence of the redox midpoint potentials of Q A ( E m(Q A) ) and Q B ( E m(Q B) ) from the proton uptake, we used a thermodynamic (model-independent) relation. E m(Q A) decreased approx. 20 mV/pH at 6.0 < pH < 10.5, while E (Q B) decreased approx. 20 mV/pH at 6.0 < pH < 8.5 and approx. 40 mV/pH at pH < 6.0 and pH > 9.0. The pH dependence of E m(Q A) in isolated reaction centers is significantly weaker than that determined from redox titrations of Q A in chromatophores of Rb sphaeroides (see for example Prince, R.C. and Dutton, P.L. (1976) Arch. Biochem. Biophys. 172, 329–334). The pH-dependence of the free energy between Q A −Q B and Q AQ B − obtained from the difference of E m(Q A) and E m(Q B) is in good agreement with that determined from the measurement of electron-transfer kinetics in isolated RCs from Rb. sphaeroides (Kleinfeld, D., Okamura, M.Y., and Feher, G. (1984) Biochim. Biophys. Acta 766, 126–140). The stronger average interaction of the protons with Q B − provides the driving force for the forward electron transfer. A simplified model was used to calculate the p K shifts from the electrostatic energy between Q A − or Q B − and the charges of the protonatable amino acid residues whose positions were obtained from the three-dimensional structure (Allen, J.P., Feher, G., Yeates, T.O., Komiya, H. and Rees, D.C. (1987) Proc. Natl. Acad. Sci. USA 84, 6162–6166).
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