TYROSINE-162 OF THE PHOTOSYNTHETIC REACTION-CENTER L-SUBUNIT PLAYS A CRITICAL ROLE IN THE CYTOCHROME-C(2) 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 isol ated 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) were 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 equimo lar RC and cyt c2 concentrations, the kinetics were very similar to th ose measured in intact cells (Farchaus et al., 1993). The fast compone nt 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 dis tinguish the contributions of association, reorientation, and electron -transfer reactions to the observed kinetics. In competition experimen ts, WTRC (L162Y) and the mutant RC L162L showed similar affinity for c yt c2, with a dissociation constant of k(D) = 10(-6) M. Mutants with a n aliphatic substitution at position L162 displayed slower cyt c2-RC a ssociation and dissociation rates. Comparison of the major kinetic com ponent of the P+ rereduction rates for the aliphatic substitutions to the aromatic substitution, L162F, revealed that the former were less a ffected by ionic strength and viscosity than the latter. The viscosity and ionic strength dependences noted for L162F were comparable to tho se seen for the slow kinetic component observed for the WT RC. The red ox 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 DELT AG not large enough to account for the drastic kinetic effects. Rather , the results suggested that the state(s) where cyt C2 is nonproductiv ely bound to the RC dominated in the mutants. In the L162F mutant, it appeared that only the distribution between the bound cyt c2 states wa s affected, whereas for the mutants with aliphatic substitutions, a de creased reorientation rate had to be additionally assumed in order to explain the observations.