Two specific domains of the γ subunit of chloroplast F _oF ₁ provide redox regulation of the ATP synthesis through conformational changes

Significance

Among the F _oF ₁-ATP synthase complexes of all organisms, chloroplast F _oF ₁ (CF _oCF ₁) is a unique enzyme with a redox regulation mechanism; however, the underlying mechanism of redox regulation of the adenosine triphosphate (ATP) synthesis reaction in CF _oCF ₁ has not been fully elucidated. By taking advantage of the powerful genetics of Chlamydomonas reinhardtii as a model organism for photosynthesis, we conducted a comprehensive biochemical analysis of the CF _oCF ₁ molecule. Here we identify structural determinants for the kinetics of the intracellular redox response and demonstrate that the redox regulation of ATP synthesis is accomplished by the cooperative interaction of two γ subunit domains of CF _oCF ₁ that are unique to photosynthetic organisms.

Abstract

Chloroplast F _oF ₁-ATP synthase (CF _oCF ₁) 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 _oCF ₁ 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 Chlamydomonas reinhardtii, we analyzed the ATP synthesis reaction and its regulation in CF _oCF ₁. The domains in the γ subunit involved in the redox regulation of CF _oCF ₁ 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 _oCF ₁ complex from C. reinhardtii 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 _oCF ₁.