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 .1. SITE-DIRECTED MUTAGENESIS AND INITIAL CHARACTERIZATION

Five site-directed mutants were engineered to substitute phenylalanine , serine, leucine, methionine, and glycine for tyrosine residue 162 of the pufL gene in Rhodobacter (R.) sphaeroides. Each of the mutations and the wild-type (WT) genes was expressed in the R. sphaeroides puf d eletion strain PUFDELTALMX21/3. Initial characterization revealed that all of the mutants were photoheterotrophically competent but that L16 2G and L162S were impaired. The amounts of mutant reaction centers exp ressed, the spectral characteristics, and the rates of intraprotein el ectron transfer and turnover were similar to the values obtained for W T. Kinetic measurements of photooxidized special pair rereduction medi ated by the physiological donor cytochrome c2 in intact chemoheterotro phically grown cells revealed that the fast phase was abolished in all mutants and that the overall kinetics of rereduction was drastically slowed. It is concluded that L162Y plays a vital role in facilitating the rapid rereduction of the photooxidized bacteriochlorophyll dimer i n R. sphaeroides.