The enzymes of the mitochondrial electron transport chain are key players of cell metabolism. Despite being active when isolated, in vivo they associate into supercomplexes¹, whose precise role is debated. Supercomplexes CIII₂CIV_1-2 (refs. ^2,3), CICIII₂ (ref. ⁴) and CICIII₂CIV (respirasome)^5,6,7,8,9,10 exist in mammals, but in contrast to CICIII₂ and the respirasome, to date the only known eukaryotic structures of CIII₂CIV_1-2 come from Saccharomyces cerevisiae^11,12 and plants¹³, which have different organization. Here we present the first, to our knowledge, structures of mammalian (mouse and ovine) CIII₂CIV and its assembly intermediates, in different conformations. We describe the assembly of CIII₂CIV from the CIII₂ precursor to the final CIII₂CIV conformation, driven by the insertion of the N terminus of the assembly factor SCAF1 (ref. ¹⁴) deep into CIII₂, while its C terminus is integrated into CIV. Our structures (which include CICIII₂ and the respirasome) also confirm that SCAF1 is exclusively required for the assembly of CIII₂CIV and has no role in the assembly of the respirasome. We show that CIII₂ is asymmetric due to the presence of only one copy of subunit 9, which straddles both monomers and prevents the attachment of a second copy of SCAF1 to CIII₂, explaining the presence of one copy of CIV in CIII₂CIV in mammals. Finally, we show that CIII₂ and CIV gain catalytic advantage when assembled into the supercomplex and propose a role for CIII₂CIV in fine tuning the efficiency of electron transfer in the electron transport chain.

线粒体电子传递链的酶是细胞代谢的关键参与者。尽管在分离时很活跃，但在体内它们会结合成超复合物¹，其确切作用尚有争议。超级复合物 CIII ₂ CIV _1-2（参考文献^2,3）、CICIII ₂（参考文献⁴）和 CICIII ₂ CIV（呼吸体）^5,6,7,8,9,10存在于哺乳动物中，但与 CICIII ₂形成对比和呼吸体，迄今为止，唯一已知的 CIII ₂ CIV _1-2真核结构来自酿酒酵母^11,12和植物¹³, 有不同的组织。在这里，我们首先介绍了哺乳动物（小鼠和绵羊）CIII ₂ CIV 及其组装中间体的不同构象结构。我们描述了 CIII ₂ CIV 从 CIII ₂前体到最终 CIII ₂ CIV 构象的组装，这是由组装因子 SCAF1（参考文献¹⁴）的 N 末端插入 CIII ₂深处驱动的，而它的 C 末端被整合进入CIV。我们的结构（包括 CICIII ₂和呼吸小体）也证实 SCAF1 是 CIII ₂ CIV 组装所必需的，并且在呼吸小体的组装中没有作用。我们表明 CIII₂是不对称的，因为仅存在一个亚基 9 拷贝，它横跨两个单体并阻止第二个 SCAF1 拷贝与 CIII ₂连接，这解释了在哺乳动物中 CIII ₂ CIV 中存在一个 CIV 拷贝。最后，我们表明 CIII ₂和 CIV 在组装成超复合物时获得催化优势，并提出 CIII ₂ CIV 在微调电子传输链中电子转移效率方面的作用。