Two specific domains of the γ subunit of chloroplast FF provide redox regulation of the ATP synthesis through conformational changes.

Chloroplast FF-ATP synthase (CFCF) converts proton motive force into chemical energy during photosynthesis. Although many studies have been done to elucidate the catalytic reaction and its regulatory mechanisms, biochemical analyses using the CFCF complex have been limited because of various technical barriers, such as the difficulty in generating mutants and a low purification efficiency from spinach chloroplasts. By taking advantage of the powerful genetics available in the unicellular green alga , we analyzed the ATP synthesis reaction and its regulation in CFCF. The domains in the γ subunit involved in the redox regulation of CFCF were mutated based on the reported structure. An in vivo analysis of strains harboring these mutations revealed the structural determinants of the redox response during the light/dark transitions. In addition, we established a half day purification method for the entire CFCF complex from and subsequently examined ATP synthesis activity by the acid-base transition method. We found that truncation of the β-hairpin domain resulted in a loss of redox regulation of ATP synthesis (i.e., constitutively active state) despite retaining redox-sensitive Cys residues. In contrast, truncation of the redox loop domain containing the Cys residues resulted in a marked decrease in the activity. Based on this mutation analysis, we propose a model of redox regulation of the ATP synthesis reaction by the cooperative function of the β-hairpin and the redox loop domains specific to CFCF.