title: Chloroplast thioredoxin mutants without active-site cysteines facilitate the reduction of the regulatory disulphide bridge on the gamma-subunit of chloroplast ATP synthase.
abstract: The activity of the chloroplast H+-ATPase (CFoCF1) is regulated by the proton electrochemical membrane potential and the reduction or the formation of the disulphide bridge on the gamma-subunit mediated by chloroplast thioredoxins (Trx). The latter regulation also applies to the water-soluble portion of CFoCF1 (CF1) and includes two successive steps, namely the binding of Trx to CF1 and the subsequent reduction or oxidation of CF1. To study this process thoroughly, a new expression system for spinach Trx-f and Trx-m was designed. In the presence of dithiothreitol (DTT) both forms of the expressed Trx could reduce the disulphide bridge on the gamma-subunit of CF1 and thus activate the ATPase. Trx mutants deficient in the internal, or both, cysteines of the active site were designed to study the details of the interaction. The Trx mutant proteins could still activate CF1-ATPase in the presence of DTT and they also increased the apparent affinity of CF1 for DTT. This implies that the binding of Trx to the CF1 gamma-subunit induces a conformational change facilitating the reduction of the disulphide bridge, and partially explains the high efficiency of Trx as a reductant in vivo.
authors.affiliation: The Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Nagatsuta 4259, Midori-Ku, Yokohama 226-8503, Japan.
authors.forename: M T
authors.initials: MT
authors.lastname: Stumpp
authors.forename: K
authors.initials: K
authors.lastname: Motohashi
authors.forename: T
authors.initials: T
authors.lastname: Hisabori
mesh_terms: D001665:Binding Sites / Q000235:genetics; D054479:Chloroplast Thioredoxins; D002736:Chloroplasts / Q000201:enzymology*; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000378:metabolism*; D004229:Dithiothreitol; D004789:Enzyme Activation; D008954:Models, Biological; D009154:Mutation; D010084:Oxidation-Reduction; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D018724:Spinacia oleracea; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*
chemical_list: D054479:Chloroplast Thioredoxins; D004220:Disulfides; D013438:Sulfhydryl Compounds; D013879:Thioredoxins; D006180:Proton-Translocating ATPases; D003545:Cysteine; D004229:Dithiothreitol