title: B-branch electron transfer in reaction centers of Rhodobacter sphaeroides assessed with site-directed mutagenesis.
abstract: Mutants of Rhodobacter (Rba.) sphaeroides are described which were designed to study electron transfer along the so-called B-branch of reaction center (RC) cofactors. Combining the mutation L(M214)H, which results in the incorporation of a bacteriochlorophyll, beta, for H(A) [Kirmaier et al. (1991) Science 251: 922-927] with two mutations, G(M203)D and Y(M210)W, near B(A), we have created a double and a triple mutant with long lifetimes of the excited state P(*) of the primary donor P, viz. 80 and 160 ps at room temperature, respectively. The yield of P(+)Q(A) (-) formation in these mutants is reduced to 50 and 30%, respectively, of that in wildtype RCs. For both mutants, the quantum yield of P(+)H(B) (-) formation was less than 10%, in contrast to the 15% B-branch electron transfer demonstrated in RCs of a similar mutant of Rba. capsulatus with a P(*) lifetime of 15 ps [Heller et al. (1995) Science 269: 940-945]. We conclude that the lifetime of P(*) is not a governing factor in switching to B-branch electron transfer. The direct photoreduction of the secondary quinone, Q(B), was studied with a triple mutant combining the G(M203)D, L(M214)H and A(M260)W mutations. In this triple mutant Q(A) does not bind to the reaction center [Ridge et al. (1999) Photosynth Res 59: 9-26]. It is shown that B-branch electron transfer leading to P(+)Q(B) (-) formation occurs to a minor extent at both room temperature and at cryogenic temperatures (about 3% following a saturating laser flash at 20 K). In contrast, in wildtype RCs P(+)Q(B) (-) formation involves the A-branch and does not occur at all at cryogenic temperatures. Attempts to accumulate the P(+)Q(B) (-) state under continuous illumination were not successful. Charge recombination of P(+)Q(B) (-) formed by B-branch electron transfer in the new mutant is much faster (seconds) than has been previously reported for charge recombination of P(+)Q(B) (-) trapped in wildtype RCs (10(5) s) [Kleinfeld et al. (1984b) Biochemistry 23: 5780-5786]. This difference is discussed in light of the different binding sites for Q(B) and Q(B) (-) that recently have been found by X-ray crystallography at cryogenic temperatures [Stowell et al. (1997) Science 276: 812-816]. We present the first low-temperature absorption difference spectrum due to P(+)Q(B) (-).
authors.affiliation: Department of Biophysics, Huygens Laboratory, Leiden University, P.O. Box 9504, 2300 RA, Leiden, The Netherlands, gast@biophys.leidenuniv.nl.
authors.forename: Arjo L
authors.initials: AL
authors.lastname: de Boer
authors.forename: Sieglinde
authors.initials: S
authors.lastname: Neerken
authors.forename: Rik
authors.initials: R
authors.lastname: de Wijn
authors.forename: Hjalmar P
authors.initials: HP
authors.lastname: Permentier
authors.forename: Peter
authors.initials: P
authors.lastname: Gast
authors.forename: Erik
authors.initials: E
authors.lastname: Vijgenboom
authors.forename: Arnold J
authors.initials: AJ
authors.lastname: Hoff