The chloroplast ATP synthase redox domain in eludes activity regulation for heterotrophic dark metabolism.

To maintain CO fixation in the Calvin-Benson-Bassham cycle, multistep regulation of the chloroplast ATP synthase (CFF) 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 CFF γ-subunit. The disulfide hampers ATP synthesis and hydrolysis reactions in dark-adapted CFF from land plants by increasing the required transmembrane electrochemical proton gradient ([Formula: see text]). Here, we show in that algal CFF 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 CFF 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 CFF, 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 CFF as the result of bioenergetic deviations from wild-type. Our results suggest that the lifestyle of requires a specific CFF dark regulation that partakes in metabolic coupling between the chloroplast and acetate-fueled mitochondria.