The second electron transfer from primary ubiquinone QA to secondary ubiquinone QB in the reaction center (RC) from Rhodobacter sphaeroides involves a protonated QB- intermediate state whose low pKa makes direct observation impossible. Here, we replaced the native ubiquinone with low-potential rhodoquinone at the QB binding site of the M265IT mutant RC. Because the in situ midpoint redox potential of QA of this mutant was lowered approximately the same extent (≈100 mV) as that of QB upon exchange of ubiquinone with low-potential rhodoquinone, the inter-quinone (QA → QB) electron transfer became energetically favorable. After subsequent saturating flash excitations, a period of two damped oscillations of the protonated rhodosemiquinone was observed. The QBH• was identified by (1) the characteristic band at 420 nm of the absorption spectrum after the second flash and (2) weaker damping of the oscillation at 420 nm (due to the neutral form) than at 460 nm (attributed to the anionic form). The appearance of the neutral semiquinone was restricted to the acidic pH range, indicating a functional pKa of <5.5, slightly higher than that of the native ubisemiquinone (pKa < 4.5) at pH 7. The analysis of the pH and temperature dependencies of the rates of the second electron transfer supports the concept of the pH-dependent pKa of the semiquinone at the QB binding site. The local electrostatic potential is severely modified by the strongly interacting neighboring acidic cluster, and the pKa of the semiquinone is in the middle of the pH range of the complex titration. The kinetic and thermodynamic data are discussed according to the proton-activated electron transfer mechanism combined with the pH-dependent functional pKa of the semiquinone at the QB site of the RC.
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