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● We solved the high-resolution structures of MCI2Ⅲ2Ⅳ2 in five conformations: State 1, State 2, Mid 1, Mid 2, and Mid 3.
● All two nicotinamide adenine dinucleotide (NADH)-ubiquinone oxidoreductases (CIs) found in constricted forms of MCI2Ⅲ2Ⅳ2 were in deactive state.
● The dynamic range of the 2Fe–2S cluster is related to the conformation of CI.
● Gated stimulated emission depletion microscopy (gSTED) was applied to verify the in vivo existence of MCI2Ⅲ2Ⅳ2.
Mammalian mitochondrial electron transport chain complexes are the most important and complicated protein machinery in mitochondria. Although this system has been studied for more than a century, its composition and molecular mechanism are still largely unknown. Here we report the high-resolution cryo-electron microscopy (Cryo-EM) structures of porcine respiratory chain megacomplex-Ⅰ2Ⅲ2Ⅳ2 (MCI2Ⅲ2Ⅳ2) in five different conformations, including State 1, State 2, Mid 1, Mid 2, and Mid 3. High-resolution Cryo-EM imaging, combined with super-resolution gated stimulated emission depletion microscopy (gSTED), strongly supports the formation of MCI2Ⅲ2Ⅳ2 in live cells. Each MCI2Ⅲ2Ⅳ2 structure contains 141 subunits (70 different kinds of peptides, 2.9 MDa) in total with 240 transmembrane helices. The mutual influence among CI, CIII, and CIV shown in the MCI2Ⅲ2Ⅳ2 structure suggests this megacomplex could act as an integral unit in electron transfer and proton pumping. The conformational changes from different states suggest a plausible regulatory mechanism for the MCI2Ⅲ2Ⅳ2 activation/deactivation process.
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