Site-Directed Mutagenesis of Reaction Centers from Rhodobacter sphaeroides
Site-directed mutagenesis is a powerful tool for studying the function of proteins. In this study we describe a genetic system for performing site-directed mutagenesis on Rhodobacter (Rb.) sphaeroides reaction centers (RCs). The RC of Rb. sphaeroides has been extensively characterized (for reviews see ref. 1 and 2) and methodologies for altering its structure have been developed (e.g. exchanging cofactors, dissociating subunits). Its three-dimensional structure has been determined by X-ray crystallography (3). Three-dimensional structural information on mutant RCs is important to distinguish between a direct effect of an altered residue and an indirect effect resulting from structural changes. A large number of RC mutants have been constructed in Rb. capsulatus, which has a well developed genetic system (4). However, at present the three-dimensional structure of the Rb. capsulatus RC has not been determined. We have used the system described here to study the mechanism of proton transfer to the reduced secondary quinone, Q B 2− , by replacing Glu-L212 with Gln and Asp. The modified characteristics of these mutant RCs are described elsewhere (5,6). Other mutations have been constructed in Rb. sphaeroides using similar systems (7,8,9).
KeywordsProton Transfer Deletion Strain Secondary Quinone Photosynthetic Growth Broad Host Range Plasmid
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