Characterization of the bonding interactions of QB upon photoreduction via A-branch or B-branch electron transfer in mutant reaction centers from Rhodobacter sphaeroides

In Rhodobacter sphaeroides reaction centers (RCs) containing the mutation Ala M260 to Trp (AM260W), transmembrane electron transfer along the full-length of the A-branch of cofactors is prevented by the loss of the QA ubiquinone, but it is possible to generate the radical pair P+HA− by A-branch electron transfer or the radical pair P+QB− by B-branch electron transfer. In the present study, FTIR spectroscopy was used to provide direct evidence for the complete absence of the QA ubiquinone in mutant RCs with the AM260W mutation. Light-induced FTIR difference spectroscopy of isolated RCs was also used to probe the neutral QB and the semiquinone QB− states in two B-branch active mutants, a double AM260W–LM214H mutant, denoted WH, and a quadruple mutant, denoted WAAH, in which the AM260W, LM214H, and EL212A–DL213A mutations were combined. The data were compared to those obtained with wild-type (Wt) RCs and the double EL212A–DL213A (denoted AA) mutant which exhibit the usual A-branch electron transfer to QB. The QB−/QB spectrum of the WH mutant is very close to that of Wt RCs indicating similar bonding interactions of QB and QB− with the protein in both RCs. The QB−/QB spectra of the AA and WAAH mutants are also closely related to one another, but are very different to that of the Wt complex. Isotope-edited IR fingerprint spectra were obtained for the AA and WAAH mutants reconstituted with site-specific 13C-labeled ubiquinone. Whilst perturbations of the interactions of the semiquinone QB− with the protein are observed in the AA and WAAH mutants, the FTIR data show that the bonding interaction of neutral QB in these two mutants are essentially the same as those for Wt RCs. Therefore, it is concluded that QB occupies the same binding position proximal to the non-heme iron prior to reduction by either A-branch or B-branch electron transfer.