The role of reaction center excited state evolution during charge separation in a Rb. sphaeroides mutant with an initial electron donor midpoint potential 260 mV above wild type

Femtosecond transient absorbance spectroscopy was performed on the triple hydrogen bond reaction center mutant [LH(L131) + LH(M160) + FH(M197)] of Rhodobacter sphaeroides which has a P/P⁺ midpoint potential 260 mV above wild type. The decay of the excited singlet state in this mutant is kinetically complex with a dominant decay component of about 50 ps at 295 K. Charge separation to the state P⁺Q_A^- occurs with a quantum yield of 0.50 ± 0.1 at 295 K and 0.10-0.15 at 20 K. The yield, rate of formation and spectra of states which are trapped when electron transfer to the quinone is blocked by quinone reduction compared to the rate and yield of formation of P⁺Q_A^- in unreduced reaction centers suggest that evolution of the excited state is the rate limiting event in charge separation in triple mutant reaction centers. The excited state that results from this evolution has spectral features which are remarkably similar to the initial excited singlet state found using R-26 reaction centers (R-26 reaction centers have essentially wild type photochemistry). The fact that the formation of this altered excited state is greatly slowed in a high P/P⁺ midpoint potential mutant suggests that the early excited state in wild type or R-26 reaction centers may have considerable P⁺ character. A consideration of the thermodynamics of the state P⁺B_A^- in this and related high potential mutants implies that a simple model in which P⁺B_A^- is formed as a discrete electron transfer intermediate is not a viable description in these mutants. Other factors such as reaction center heterogeneity or alternate electron transfer mechanisms must be invoked.