The chloroplast ATP synthase redox domain in <i>Chlamydomonas reinhardtii</i> eludes activity regulation for heterotrophic dark metabolism

To maintain CO₂ fixation in the Calvin-Benson-Bassham cycle, multistep regulation of the chloroplast ATP synthase (CF₁F_o) is crucial to balance the ATP output of photosynthesis with protection of the apparatus. A well-studied mechanism is thiol modulation; a light/dark regulation through reversible cleavage of a disulfide in the CF₁F_o γ-subunit. The disulfide hampers ATP synthesis and hydrolysis reactions in dark-adapted CF₁F_o from land plants by increasing the required transmembrane electrochemical proton gradient ([Formula: see text]). Here, we show in <i>Chlamydomonas reinhardtii</i> that algal CF₁F_o is differently regulated in vivo. A specific hairpin structure in the γ-subunit redox domain disconnects activity regulation from disulfide formation in the dark. Electrochromic shift measurements suggested that the hairpin kept wild-type CF₁F_o active, whereas the enzyme was switched off in algal mutant cells expressing a plant-like hairpin structure. The hairpin segment swap resulted in an elevated [Formula: see text] threshold to activate plant-like CF₁F_o, increased by ~1.4 photosystem (PS) I charge separations. The resulting dark-equilibrated [Formula: see text] dropped in the mutants by ~2.7 PSI charge separation equivalents. Photobioreactor experiments showed no phenotypes in autotrophic aerated mutant cultures. In contrast, chlorophyll fluorescence measurements under heterotrophic dark conditions point to an altered dark metabolism in cells with the plant-like CF₁F_o as the result of bioenergetic deviations from wild-type. Our results suggest that the lifestyle of <i>C. reinhardtii</i> requires a specific CF₁F_o dark regulation that partakes in metabolic coupling between the chloroplast and acetate-fueled mitochondria.