B side electron transfer in a rhodobacter sphaeroides reaction center mutant in which the B side monomer bacteriochlorophyll is replaced with bacteriopheophytin: Low-temperature study and energetics of charge-separated states

The mutation, HL(M182), in the Rhodobacter sphaeroides reaction center (RC) results in the replacement of the monomer bacteriochlorophyll (BChl) on the inactive side (B side) of the RC with a bacteriopheophytin (BPheo; the new cofactor is referred to as φ_B). In φ_B-containing RCs, the first excited state of the primary donor (P*) decays with an accelerated time constant of 2.6 ± 0.1 ps at room temperature as compared to 3.1 ± 0.2 ps in wild-type (WT) RCs. At low temperatures, P* decay is essentially the same in the HL(M182) mutant and WT RCs: 1.4 ± 0.1 ps at 77 K and 1.1 ± 0.1 ps at 9 K. The yield of the charge-separated P⁺φ_B^- state decreases from 35% at room temperature to 12% at 77 and 9 K. The decreased P⁺φ_B^- yield is apparently due to the fact that the rate of the charge separation along the A side of the RC at low temperature increases, while the rate along the B side remains essentially unchanged. From measurements of the long-lived fluorescence decay at room temperature, the standard free energy of the P⁺φ_B^- state is estimated to be about 0.16 ± 0.04 eV below P*. Given a difference between the midpoint potentials of BChl and BPheo of 0.26 ± 0.03 V, the standard free energy of the P⁺B_B^- state in WT RC is estimated to be 0.1 ± 0.07 eV above P*.