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. 1. Site-directed mutagenesis and initial characterization

Abstract

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 deletion strain PUF delta LMX21/3. Initial characterization revealed that all of the mutants were photoheterotrophically competent but that L162G and L162S were impaired. The amounts of mutant reaction centers expressed, the spectral characteristics, and the rates of intraprotein electron transfer and turnover were similar to the values obtained for WT. Kinetic measurements of photooxidized special pair rereduction mediated by the physiological donor cytochrome c2 in intact chemoheterotrophically 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 in R. sphaeroides.