Primary processes of charge separation in reaction centers of YM210L/FM197Y and YM210L mutants of Rhodobacter sphaeroides.

Difference femtosecond absorption spectroscopy with 20-fsec temporal resolution was applied to study a primary stage of charge separation and transfer processes in reaction centers of YM210L and YM210L/FM197Y site-directed mutants of the purple bacterium Rhodobacter sphaeroides at 90 K. Photoexcitation was tuned to the absorption band of the primary electron donor P at 880 nm. Coherent oscillations in the kinetics of stimulated emission of P* excited state at 940 nm and of anion absorption of monomeric bacteriochlorophyll B(A)(-) at 1020 nm were monitored. The absence of tyrosine YM210 in RCs of both mutants leads to strong slowing of the primary reaction P* --> P(+)B(A)(-) and to the absence of stabilization of separated charges in the state P(+)B(A)(-). Mutation FM197Y increases effective mass of an acetyl group of pyrrole ring I in the bacteriochlorophyll molecule P(B) of the double mutant YM210L/FM197Y by a hydrogen bond with OH-TyrM197 group that leads to a decrease in the frequency of coherent nuclear motions from 150 cm(-1) in the single mutant YM210L to ~100 cm(-1) in the double mutant. Oscillations with 100-150 cm(-1) frequencies in the dynamics of the P* stimulated emission and in the kinetics of the reversible formation of P(+)B(A)(-) state of both mutants reflect a motion of the P(B) molecule relatively to P(A) in the area of mutual overlapping of their pyrrole rings I. In the double mutant YM210L/FM197Y the oscillations in the P* emission band and the B(A)(-) absorption band are conserved within a shorter time ~0.5 psec (1.5 psec in the YM210L mutant), which may be a consequence of an increase in the number of nuclei forming a wave packet by adding a supplementary mass to the dimer P.