To maintain CO 2 fixation in the Calvin–Benson–Bassham cycle, multistep regulation of the chloroplast ATP synthase (CF 1 F 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 CF 1 F o γ-subunit. The disulfide hampers ATP synthesis and hydrolysis reactions in dark-adapted CF 1 F o from land plants by increasing the required transmembrane electrochemical proton gradient ( Δ μ ∼ H + ). Here, we show in Chlamydomonas reinhardtii that algal CF 1 F 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 CF 1 F 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 Δ μ ∼ H + threshold to activate plant-like CF 1 F o , increased by ~1.4 photosystem (PS) I charge separations. The resulting dark-equilibrated Δ μ ∼ H + 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 CF 1 F o as the result of bioenergetic deviations from wild-type. Our results suggest that the lifestyle of C. reinhardtii requires a specific CF 1 F o dark regulation that partakes in metabolic coupling between the chloroplast and acetate-fueled mitochondria.

中文翻译：

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莱茵衣藻中的叶绿体 ATP 合酶氧化还原结构域逃避了异养暗代谢的活性调节


为了在 Calvin-Benson-Bassham 循环中维持 CO 2 固定，叶绿体 ATP 合酶 （CF 1 F o） 的多步调节对于平衡光合作用的 ATP 输出与设备的保护至关重要。一个经过充分研究的机制是硫醇调制;通过 CF 1 F o γ 亚基中二硫键的可逆裂解进行明/暗调节。二硫键通过增加所需的跨膜电化学质子梯度 （Δ μ ∼ H +） 来阻碍陆地植物暗适应 CF 1 F o 中的 ATP 合成和水解反应。在这里，我们在莱茵衣藻中表明，藻类 CF 1 F o 在体内受到不同的调节。γ 亚基氧化还原结构域中的特异性发夹结构将活性调节与黑暗中二硫键形成断开。电致变色偏移测量表明，发夹保持野生型 CF 1 F o 活性，而酶在表达植物样发夹结构的藻类突变细胞中被关闭。发夹段交换导致 Δ μ升高 ∼ H + 阈值以激活植物样 CF 1 F o，增加 ~1.4 光系统 （PS） I 电荷分离。所得的暗平衡 Δ μ ∼ H + 在突变体中下降了 ~2.7 PSI 电荷分离当量。光生物反应器实验显示，自养曝气突变体培养物中没有表型。相比之下，异养黑暗条件下的叶绿素荧光测量表明，由于生物能量偏离野生型，具有植物样 CF 1 F o 的细胞中的暗代谢发生了变化。我们的结果表明，C. reinhardtii 的生活方式需要特定的 CF 1 F o 暗调节，该调节参与叶绿体和乙酸盐燃料线粒体之间的代谢耦合。