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(+)Q(B)(-) formation via the B-branch. The A-branch of the mutant RC contains two monomer bacteriochlorophylls, 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(+)Q(B)(-) 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 (approximately 1%) P(+)Q(B)(-) formation was found.