The respiratory complexes are organized in supramolecular assemblies called supercomplexes thought to optimize cellular metabolism under physiological and pathological conditions. In this study, we used genetically and biochemically well characterized cells bearing the pathogenic microdeletion m.15,649-15,666 (ΔI300-P305) in MT-CYB gene, to investigate the effects of an assembly-hampered CIII on the re-organization of supercomplexes. First, we found that this mutation also affects the stability of both CI and CIV, and evidences the occurrence of a preferential structural interaction between CI and CIII2, yielding a small amount of active CI+CIII2 supercomplex. Indeed, a residual CI+CIII combined redox activity, and a low but detectable ATP synthesis driven by CI substrates are detectable, suggesting that the assembly of CIII into the CI+CIII2 supercomplex mitigates the detrimental effects of MT-CYB deletion. Second, measurements of oxygen consumption and ATP synthesis driven by NADH-linked and FADH2-linked substrates alone, or in combination, indicate a common ubiquinone pool for the two respiratory pathways. Finally, we report that prolonged incubation with rotenone enhances the amount of CI and CIII2, but reduces CIV assembly. Conversely, the antioxidant N-acetylcysteine increases CIII2 and CIV2 and partially restores respirasome formation. Accordingly, after NAC treatment, the rate of ATP synthesis increases by two-fold compared with untreated cell, while the succinate level, which is enhanced by the homoplasmic mutation, markedly decreases. Overall, our findings show that fine-tuning the supercomplexes stability improves the energetic efficiency of cells with the MT-CYB microdeletion.

中文翻译：

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在复合物 III 缺陷的细胞中微调呼吸复合物的稳定性和超级复合物的组装。

呼吸复合物被组织成称为超级复合物的超分子组装体，被认为可以优化生理和病理条件下的细胞代谢。在这项研究中，我们使用在 MT-CYB 基因中带有致病性微缺失 m.15,649-15,666 (ΔI300-P305) 的遗传和生化特征良好的细胞，研究组装受阻 CIII 对超级复合物重组的影响。首先，我们发现这种突变也会影响 CI 和 CIV 的稳定性，并证明 CI 和 CIII2 之间发生了优先结构相互作用，产生了少量的活性 CI+CIII2 超复合物。事实上，残留的 CI+CIII 组合氧化还原活性，以及​​由 CI 底物驱动的低但可检测的 ATP 合成是可检测的，表明将 CIII 组装到 CI+CIII2 超复合物中减轻了 MT-CYB 缺失的不利影响。其次，由单独或组合的 NADH 连接和 FADH2 连接底物驱动的耗氧量和 ATP 合成的测量表明，这两种呼吸途径有一个共同的泛醌池。最后，我们报告说，与鱼藤酮的长时间孵育会增加 CI 和 CIII2 的数量，但会减少 CIV 组装。相反，抗氧化剂 N-乙酰半胱氨酸增加 CIII2 和 CIV2 并部分恢复呼吸体形成。因此，在 NAC 处理后，与未处理细胞相比，ATP 合成速率增加了两倍，而由同质突变增强的琥珀酸水平显着降低。总体，