Tyrosine 162 of the photosynthetic reaction center L-subunit plays a critical role in the cytochrome c2 mediated rereduction of the photooxidized bacteriochlorophyll dimer in Rhodobacter sphaeroides. 2. Quantitative kinetic analysis.

The electron-transfer kinetics from the soluble cytochrome (cyt) c2 to the photooxidized reaction center (RC) was studied with proteins isolated from Rhodobacter (R.) sphaeroides. In addition to wild-type (WT) RC, RCs harboring site-directed mutations at residue L162 (L162F, -M, -L, -S, or -G) wree analyzed. The disappearance of the absorption band of the photooxidized primary donor P+ (at 1250 nm) and the alpha-band of cyt c2 (at 550 nm) were monitored. Under conditions of high equimolar RC and cyt c2 concentrations, the kinetics were very similar to those measured in intact cells (Farchaus et al., 1993). The fast component of the kinetics normally seen in WT was not observed in any of the mutants; the overall rereduction rates for the mutants depended on the amino acid substitution. Light intensity, viscosity, ionic strength, and RC/cyt c2 stoichiometry of the reaction mixture were varied to distinguish the contributions of association, reorientation, and electron-transfer reactions to the observed kinetics. In competition experiments, WTRC (L162Y) and the mutant RCL162L showed similar affinity for cyt c2, with a dissociation constant of kD = 10(-6) M. Mutants with an aliphatic substitution at position L162 displayed slower cyt c2-RC association and dissociation rates. Comparison of the major kinetic component of the P+ rereduction rates for the aliphatic substitutions to the aromatic substitution, L162F, revealed that the former were less affected by ionic strength and viscosity than the latter. The viscosity and ionic strength dependences noted for L162F were comparable to those seen for the slow kinetic component observed for the WT RC. The redox midpoint potential of the P/P+ couple was increased by 30 mV (L162F) to 50 mV (L162L, G) over the WT value, leading to differences in delta G not large enough to account for the drastic kinetic effects. Rather, the results suggested that the state(s) where cyt c2 is nonproductively bound to the RC dominated in the mutants. In the L162F mutant, it appeared that only the distribution between the bound cyt c2 states was affected, whereas for the mutants with aliphatic substitutions, a decreased reorientation rate had to be additionally assumed in order to explain the observations.