Chloroplast ATP synthase is a thiol-modulated enzyme whose ∆µH+-linked activation is strongly influenced by reduction and the formation of a disulphide bridge between Cys199 and Cys205 on the γ subunit. In solubilized chloroplast coupling factor 1 (CF1), reduction of the disulphide bond elicits the latent ATP-hydrolysing activity. To assess the regulatory importance of the amino acid residues around these cysteine residues, we focused on the three negatively charged residues Glu210-Asp-Glu212 close to the two cysteine residues and also on the following region from Leu213 to Ile230, and investigated the modulation of ATPase activity by chloroplast thioredoxins. The mutant γ subunits were reconstituted with the α and β subunits from F1 of the thermophilic bacterium Bacillus PS3; the active ATPase complexes obtained were purified by gel-filtration chromatography. The complex formed with a mutant γ subunit in which Glu210 to Glu212 had been deleted was inactivated rather than activated by reduction of the disulphide bridge by reduced thioredoxin, indicating inverse regulation. This complex was insensitive to the inhibitory CF1-ε subunit when the mutant γ subunit was oxidized. In contrast, the deletion of Glu212 to Ile230 converted the complex from a modulated state into a highly active state.

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