The unusual chloroplast ATP synthase redox domain ensures enzyme activity and elevates the electrochemical proton gradient in dark-adapted Chlamydomonas reinhardtii.

To maintain CO2 fixation in the Calvin Benson-Bassham cycle, multi-step regulation of the chloroplast ATP synthase (CF1Fo) 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 CF1Fo {gamma}-subunit. The disulfide hampers ATP synthesis and hydrolysis reactions in dark-adapted CF1Fo from land plants by increasing the required transmembrane electrochemical proton gradient [Formula]. Here, we show in Chlamydomonas reinhardtii that algal CF1Fo is differently regulated in vivo. A specific hairpin structure in the {gamma}-subunit redox domain disconnects activity regulation from disulfide formation in the dark. Electrochromic shift measurements suggested that the hairpin kept wild type CF1Fo 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] threshold to activate plant-like CF1Fo, increased by [~]1.4 photosystem (PS) I charge separations. The resulting dark-equilibrated [Formula] 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 a reduced plastoquinone pool in cells with the plant-like CF1Fo as the result of bioenergetic bottlenecks. Our results suggest that the lifestyle of Chlamydomonas reinhardtii requires a specific CF1Fo dark regulation that partakes in metabolic coupling between the chloroplast and acetate-fueled mitochondria. Significance StatementThe microalga Chlamydomonas reinhardtii exhibits a non-classical thiol modulation of the chloroplast ATP synthase for the sake of metabolic flexibility. The redox switch, although established, was functionally disconnected in vivo thanks to a hairpin segment in the {gamma}-subunit redox domain. Dark enzymatic activity was prevented by replacing the algal hairpin segment with the one from land plants, restoring a classical thiol modulation pattern. Thereby, ATP was saved at the expense of thylakoid membrane energization levels in the dark. However, metabolism was impaired upon silencing dark ATPase activity, indicating that a functional disconnect from the redox switch represents an adaptation to different ecological niches.