High yield of B-branch electron transfer in a quadruple reaction center mutant of the photosynthetic bacterium Rhodobacter sphaeroides

A new reaction center (RC) quadruple mutant, called LDHW, of Rhodobacter sphaeroides is described. This mutant was constructed to obtain a high yield of B-branch electron transfer and to study P(+)QB(-) formation via the B-branch. The A-branch of the mutant RC contains two monomer bacterio-chlorophylls, B-A and beta, as a result of the H mutation L(M214)H. The latter bacteriochlorophyll replaces bacteriopheophytin H-A of wild-type RCs. As a result of the W mutation A(M260)W, the A-branch does not contain the ubiquinone Q(A); this facilitates the study Of P(+)QB(-) formation. Furthermore, the D mutation G(M203)D introduces an aspartic acid residue near B-A. Together these mutations impede electron transfer through the A-branch. The B-branch contains two bacteriopheophytins, Phi(B) and H-B, and a ubiquinone, Q(B). Phi(B) replaces the monomer bacteriochlorophyll B-B as a result of the L mutation H(M182)L. In the LDHW mutant we find 35-45% B-branch electron transfer, the highest yield reported so far. Transient absorption spectroscopy at 10 K, where the absorption bands due to the Q(X) transitions of Phi(B) and H-B are well resolved, shows simultaneous bleachings of both absorption bands. Although photoreduction of the bacteriopheophytins occurs with a high yield, no significant (similar tol%) P(+)QB(-) formation was found.