Chloroplast thioredoxin mutants without active-site cysteines facilitate the reduction of the regulatory disulphide bridge on the γ-subunit of chloroplast ATP synthase

The activity of the chloroplast H⁺-ATPase (CF_oCF₁) is regulated by the proton electrochemical membrane potential and the reduction or the formation of the disulphide bridge on the γ-subunit mediated by chloroplast thioredoxins (Trx). The latter regulation also applies to the water-soluble portion of CF_oCF₁ (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 γ-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 γ-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.