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bioRxiv · 10.1101/2020.04.28.066100

ECS-based investigation of chloroplast ATP synthase regulation

Buchert, F.·Bailleul, B.·Joliot, P.
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Abstract

The chloroplast ATP synthase (CF1Fo) contains a specific feature to the green lineage: a {gamma}-subunit redox domain which contains a cysteine couple and interacts with the torque-generating {beta}DELSEED-loop. Based on the recently solved structure of this domain, it was proposed to function as a chock. In vitro, {gamma}-disulfide formation slows down the activity of the CF1Fo at low transmembrane electrochemical proton gradient [Formula]. Here, we utilize in vivo absorption spectroscopy measurements for functional CF1Fo activity characterization in Arabidopsis leaves. The spectroscopic method allows us to measure the [Formula] present in dark-adapted leaves, and to identify its mitochondrial sources. Furthermore, we follow the fate of the extra [Formula] generated by an illumination, including its osmotic and electric components, and from there we estimate the lifetime of the light-generated ATP. In contrast with a previous report [Joliot and Joliot, Biochim. Biophys. Acta, 1777 (2008) 676-683], the CF1Fo {gamma}-subunit exists mostly in an oxidized form in the dark-adapted state. To study the redox regulation of the CF1Fo, we used thiol agent infiltration in WT and a mutant that does not form the {gamma}-disulfide. The obtained [Formula] -dependent CF1Fo activity profile in the two {gamma}-redox states in vivo reconciles with previous biochemical in vitro findings [Junesch and Graber, Biochim. Biophys. Acta, 893 (1987) 275-288]. The highest rates of ATP synthesis we measured in the two {gamma}-redox state were similar at high [Formula]. In the presence of the {gamma}-dithiol, similar rates were obtained at a ~45 mV lower [Formula] value compared to the oxidized state, which closely resembled the energetic gap of 0.7 {Delta}pH units reported in vitro.

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Buchert, F., Bailleul, B., Joliot, P.. 2020-04-29. ECS-based investigation of chloroplast ATP synthase regulation. https://doi.org/10.1101/2020.04.28.066100

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