Site-Directed Mutagenesis of Reaction Centers from Rhodobacter sphaeroides

Abstract

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).