Two specific domains of the γ subunit of chloroplast F_oF₁ provide redox regulation of the ATP synthesis through conformational changes.

Abstract

Chloroplast F_oF₁-ATP synthase (CF_oCF₁) 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 CF_oCF₁ 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 <i>Chlamydomonas reinhardtii</i>, we analyzed the ATP synthesis reaction and its regulation in CF_oCF₁. The domains in the γ subunit involved in the redox regulation of CF_oCF₁ 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 CF_oCF₁ complex from <i>C. reinhardtii</i> 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 CF_oCF₁.