Primary electron transfer in membrane-bound reaction centers with mutations at the M210 position

The kinetics of primary electron transfer in membrane-bound Rhodobacter sphaeroides reaction centers (RCs) were measured on both wild type (WT) and site-directed mutant RC's bearing mutations at the tyrosine M210 position. The tyrosine was replaced by histidine (H), phenylalanine (F), leucine (L), or tryptophan (W). A mutant with histidine at both the M210 and symmetry-related L181 positions (YM210H/FL181H) was also examined. Rates of primary charge separation were determined by both single and multiple wavelength pump-probe techniques. The time constants for the decay of stimulated emission in the membrane-bound mutant RC's were approximately 27 ps (F), 36 ps (L), 72 ps (W), 5.8 ps (H), and 4.2 ps (HH), compared with 4.6 ps in WT membrane-bound RC's. For all RC's, the decay of stimulated emission was found to be multiexponential, demonstrating that this phenomenon is not a consequence of the removal of the RC from the membrane. The source of the multiexponential decay of the primary donor excited state was examined, leading to the conclusion that a distribution in the driving force (AG) for electron transfer cannot be the sole parameter that determines the multiexponential character. Further measurements on membrane-bound mutant RC's showed that chemical prereduction of the acceptor quinones resulted in a significant slowing of the rate of primary charge separation. This was most marked in those mutants in which the rate of charge separation had already been slowed down as a result of mutagenesis at the M210 position. The phenomenon is discussed in terms of the Coulombic interaction between Q(A)(-) and the other pigments involved in electron transfer and the influence of this interaction on the driving force for charge separation.