Mitochondrial respiratory complexes assemble into supercomplexes (SC). Q-respirasome (III2 + IV) requires the supercomplex assembly factor (SCAF1) protein. The role of this factor in the N-respirasome (I + III2 + IV) and the physiological role of SCs are controversial. Here, we study C57BL/6J mice harboring nonfunctional SCAF1, the full knockout for SCAF1, or the wild-type version of the protein and found that exercise performance is SCAF1 dependent. By combining quantitative data–independent proteomics, 2D Blue native gel electrophoresis, and functional analysis of enriched respirasome fractions, we show that SCAF1 confers structural attachment between III2 and IV within the N-respirasome, increases NADH-dependent respiration, and reduces reactive oxygen species (ROS). Furthermore, the expression of AOX in cells and mice confirms that CI-CIII superassembly segments the CoQ in two pools and modulates CI-NADH oxidative capacity.

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25Mitochondrial respiratory complexes assemble into different forms of supercomplexes 26 (SC). In particular, SC III2+IV require the SCAF1 protein. However, the structural role of this 27 factor in the formation of the respirasome (I+III2+IV) and the physiological role of SCs are 28 controversial. Here, we study C57BL/6J mice harbouring either non-functional SCAF1, the full 29 knock-out for SCAF1 or the wild-type version of the protein and found a growth and exercise 30 phenotype due to the lack of functional SCAF1. By combining quantitative data-independent 31 proteomics, high resolution 2D Blue Native Gel Electrophoresis and functional analysis of 32 enriched respirasome fractions, we show that SCAF1 confers structural attachment between III2 33 and IV within the respirasome, increases NADH-dependent respiration and reduces ROS 34 production. Furthermore, through the expression of AOX in cells and mice we confirm that CI-35 CIII superassembly segments the CoQ in two pools and modulates CI-NADH oxidative capacity. 36These data demonstrate that SC assembly, regulated by SCAF1, modulates the functionality of 37 the electron transport chain. 38

The mitochondrial electron transport chain (mETC) converts the energy of substrate oxidation into a H+ electrochemical gradient (Δp), which is composed by an inner mitochondrial membrane (IMM) potential (ΔΨmt) and a pH gradient (ΔpH). So far, ΔΨmt has been assumed to be composed exclusively by H+. Mitochondrial Ca2+ and Na+ homeostasis, which are essential for cellular function, are controlled by exchangers and antiporters in the inner mitochondrial membrane (IMM). In the last few years, some of them have been identified, except for the mitochondrial Na+/H+ exchanger (mNHE). Here, using a rainbow of mitochondrial and nuclear genetic models, we have identified it as, specifically, the P-module of complex I (CI). In turn, its activity creates a Na+ gradient across the IMM, parallel to ΔpH, which accounts for half of the ΔΨmt in coupled respiring mitochondria. We have also found that a deregulation of this mNHE function in CI, without affecting its enzymatic activity, occurs in Leber hereditary optic neuropathy (LHON), which has profound consequences in ΔΨmt and mitochondrial Ca2+ homeostasis and explains the previously unknown molecular pathogenesis of this neurodegenerative disease.