Inverse regulation of F1-ATPase activity by a mutation at the regulatory region on the γ subunit of chloroplast ATP synthase

摘要

pChloroplast ATP synthase is a thiol-modulated enzyme whose ?iμ/iHsup+/sup-linked activation is strongly influenced by reduction and the formation of a disulphide bridge between Cyssup199/sup and Cyssup205/sup on the γ subunit. In solubilized chloroplast coupling factor 1 (CFsub1/sub), 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 Glusup210/sup-Asp-Glusup212/sup close to the two cysteine residues and also on the following region from Leusup213/sup to Ilesup230/sup, and investigated the modulation of ATPase activity by chloroplast thioredoxins. The mutant γ subunits were reconstituted with the α and β subunits from Fsub1/sub of the thermophilic bacterium iBacillus/i PS3; the active ATPase complexes obtained were purified by gel-filtration chromatography. The complex formed with a mutant γ subunit in which Glusup210/sup to Glusup212/sup 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 CFsub1/sub-ε subunit when the mutant γ subunit was oxidized. In contrast, the deletion of Glusup212/sup to Ilesup230/sup converted the complex from a modulated state into a highly active state./p