作者: Dominique A. Glauser , Florence Bourquin , Wanda Manieri , Peter Schürmann
关键词: Chemistry 、 Biochemistry 、 Mutant 、 Active site 、 Site-directed mutagenesis 、 Wild type 、 Thioredoxin 、 Ferredoxin-thioredoxin reductase 、 Ferredoxin 、 Thioredoxin reductase
摘要: Ferredoxin:thioredoxin reductase (FTR) is a key regulatory enzyme of oxygenic photosynthetic cells involved in the reductive regulation important target enzymes. It catalyzes two-electron reduction disulfide thioredoxins with electrons from ferredoxin involving 4Fe-4S cluster and an adjacent active-site disulfide. We replaced Cys-57, Cys-87, His-86 active site Synechocystis FTR by site-directed mutagenesis studied properties mutated proteins. Mutation either cysteines yields inactive enzymes, which have different spectral properties, indicating reduced Fe-S when inaccessible Cys-87 oxidized accessible Cys-57 replaced. The latter mutant can be reversibly dithionite showing that it functional. C57S very stable protein, whereas C87A more labile because missing interaction cluster. replacement greatly reduces its catalytic activity supporting proposal increases nucleophilicity neighboring cysteine. Ferredoxin forms non-covalent complexes wild type (WT) FTRs, are except mutant. WT FTRs form covalent heteroduplexes modified thioredoxins. In particular, formed represent interesting one-electron-reduced reaction intermediates, split Heteroduplexes demonstrating ability to simultaneously dock thioredoxin ferredoxin, accord proposed mechanism structural analyses.