Structural analysis of the regulatory dithiol-containing domain of the chloroplast ATP synthase gamma subunit.
The gamma subunit of the F1 portion of the chloroplast ATP synthase contains a critically placed dithiol that provides a redox switch converting the enzyme from a latent to an active ATPase. The switch prevents depletion of intracellular ATP pools in the dark when photophosphorylation is inactive. The dithiol is located in a special regulatory segment of about 40 amino acids that is absent from the gamma subunits of the eubacterial and mitochondrial enzymes. Site-directed mutagenesis was used to probe the relationship between the structure of the gamma regulatory segment and its function in ATPase regulation via its interaction with the inhibitory epsilon subunit. Mutations were designed using a homology model of the chloroplast gamma subunit based on the analogous structures of the bacterial and mitochondrial homologues. The mutations included (a) substituting both of the disulfide-forming cysteines (Cys199 and Cys205) for alanines, (b) deleting nine residues containing the dithiol, (c) deleting the region distal to the dithiol (residues 224-240), and (d) deleting the entire segment between residues 196 and 241 with the exception of a small spacer element, and (e) deleting pieces from a small loop segment predicted by the model to interact with the dithiol domain. Deletions within the dithiol domain and within parts of the loop segment resulted in loss of redox control of the ATPase activity of the F1 enzyme. Deleting the distal segment, the whole regulatory domain, or parts of the loop segment had the additional effect of reducing the maximum extent of inhibition obtained upon adding the epsilon subunit but did not abolish epsilon binding. The results suggest a mechanism by which the gamma and epsilon subunits interact with each other to induce the latent state of the enzyme.