Observation of the protonated semiquinone intermediate in isolated reaction centers from Rhodobacter sphaeroides:: Implications for the mechanism of electron and proton transfer in proteins

A proton-activated electron transfer (PAET) mechanism, involving a protonated semiquinone intermediate state, had been proposed for the electron-transfer reaction k(AB)((2)) [Q(A) (-.)Q(B)(-.) + H+ reversible arrow Q(A)(-.)(Q(B)H)(.) --> Q(A)(Q(B)H)(-)] in reaction centers (RCs) from Rhodobacter sphaeroides [Graige, M. S., Paddock, M. L., Bruce, M. L., Feher, G., and Okamura, M. Y. (1996) J. Am. Chem, Soc. 118, 9005-9016], Confirmation of this mechanism by observing the protonated semiquinone (Q(B)H)(.) had not been possible, presumably because of its low pK(a). By replacing the native Q(10) in the Q(B) site with rhodoquinone (RQ), which has a higher pK(a), we were able to observe the (Q(B)H)(.) state. The pH dependence of the semiquinone optical spectrum gave a pK(a) = 7.3 +/- 0.2. At pH < pK(a), the observed rate for the k(AB)((2)) reaction was constant and attributed to the intrinsic electron-transfer rate from Q(A)(-.) to the protonated semiquinone (i.e., k(AB)((2)) = k(ET)(RQ) = 2 X 10(4) s(-1)). The rate decreased at pH > pK(a) as predicted by the PAET mechanism in which fast reversible proton transfer precedes rate-limiting electron transfer. Consequently, near pH 7, the proton-transfer rate k(H) >> 10(4) s(-1). Applying the two step mechanism to RCs containing native Q(10) and taking into account the change in redox potential, we find reasonable values for the fraction of (Q(B)H)(.) congruent-to 0.1% (consistent with a pK(a)(Q(10)) of similar-to-4.5) and k(ET)(Q(10)) congruent to 10(6) s(-1). These results confirm the PAET mechanism in RCs with RQ and give strong support that this mechanism is active in RCs with Q(10) as well.