STUDY OF WILD-TYPE AND GENETICALLY-MODIFIED REACTION CENTERS FROM RHODOBACTER-CAPSULATUS - STRUCTURAL COMPARISON WITH RHODOPSEUDOMONAS-VIRIDIS AND RHODOBACTER-SPHAEROIDES

Reaction centers from the purple bacterium Rhodobacter (Rb.) capsulatus and from two mutants Thr(L226) --> Ala and Ile(L229) --> Ser, modified in the binding protein pocket of the secondary quinone acceptor (Q(B)), have been studied by flash-induced absorbance spectroscopy. In Thr(L226) --> Ala, the binding affinities for endogenoUS QB (ubiquinone 10) and UQ6 are found to be two to three times as high as in the wild type. In contrast, in Ile(L229) --> Ser, the binding affinity for UQ6 is decreased about three times compared to the wild type. In Thr(L226) --> Ala, a markedly increased sensitivity (approximately 30 times) to o-phenanthroline is observed. In Rhodopseudomonas viridis, where Ala is naturally in position L226, the sensitivity to o-phenanthroline is close to that observed in Thr(L226) --> Ala. We propose that the presence of Ala in position L226 is responsible for the high sensitivity to that inhibitor. The pH dependencies of the rate constants of P+Q(B-)(k(BP)) charge recombination kinetics (P is a dimer of bacteriochlorophyll, and Q(B) is the secondary quinone electron acceptor) show destabilization of Q(B)- in Thr(L226) --> Ala and Ile(L229) --> Ser, compared to the wild type. At low pH, similar apparent pK values of protonation of amino acids around Q(B-) are measured in the wild type and the mutants. In contrast to Rb. sphaeroides, in the wild type Rb. capsulatus, k(BP) substantially increases in the pH range 7-1 0. This may reflect some differences in the respective structures of both strains or, alternatively, may be due to deprotonation of Tyr(L215) in Rb. capsulatus. At pH 7, measurements of the rate constant of Q(A)- to Q(B) electron transfer reveal a threefold greater rate in the reaction centers from wild type Rb. capsulatus (65 +/- 1 0 mus)-1 compared to Rb. sphaeroides. We suggest that this may arise from a 0.7-angstrom smaller distance between the quinones in the former strain. Our spectroscopic data on the wild type Rb. capsulatus reaction center suggest the existence of notable differences with the Rb. sphaeroides reaction center structure.