THE NATURE AND DYNAMICS OF THE CHARGE-SEPARATED INTERMEDIATE IN REACTION CENTERS IN WHICH BACTERIOCHLOROPHYLL REPLACES THE PHOTOACTIVE BACTERIOPHEOPHYTIN .2. THE RATES AND YIELDS OF CHARGE SEPARATION AND RECOMBINATION

The primary photochemistry of the (M)L214H and (M)L214H/(L)E104V mutant bacterial reaction centers (RCs) from Rhodobacter sphaeroides has been investigated at room and cryogenic temperatures. In both mutants the native bacteriopheophytin electron acceptor (BPh(L)) is replaced with a bacteriochlorophyll (BChl) molecule denoted by beta; in the double mutant a hydrogen-bonding interaction of beta is removed. The initial stage of charge separation, formation of an intermediate P+I-, is slowed somewhat in both mutants but without a detectable loss in yield. However, the yield of the subsequent stage of charge separation, P+I- --> P(+)QA(-), is significantly reduced due to the combination of slower electron transfer from I- to Q(A) and enhanced charge recombination of P+I- to the ground state. For example, in the double mutant the inherent time constant for electron transfer at 285 K is lengthened 10-fold to similar to 2 ns and the inherent time constant for charge recombination is reduced 20-fold to similar to 750 ps, giving a p(+)Q(A)(-) yield of 27%. At 77 K, in both beta-containing RCs electron transfer from I- to Q(A) is slowed and the p(+)Q(A)(-) yield reduced compared to the value at 285 K. The kinetic data and the spectroscopic results presented in the preceding article combine to require a substantial involvement of P(+)BChl(L)(-) in defining the character and dynamics of P+I- in the beta-type RCs. Models are considered in which P(+)beta(-) and p(+)BChl(L)(-) are quantum-mechanically mixed or in thermal equilibrium. It is concluded that P(+)beta(-) and p(+)BChl(L)(-) are very close in energy in the mutants and that P(+)BChI(L)(-) is very close in energy to the primary electron donor, P.*, in both the mutant and wild-type RCs. We also propose that the energy of p(+)BChl(L)(-) is important not only in determining the dynamics of the initial stage of charge separation but also in dictating the rates and yields of the subsequent electron transfer and charge recombination processes.