Protonated Rhodosemiquinone at the QB Binding Site of the M265IT Mutant Reaction Center of Photosynthetic Bacterium Rhodobacter sphaeroides

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 t...