Mitochondrial ATP production is achieved using the electron transport chain (ETC), whereby the controlled oxidation of biomolecules is coupled to the activity of ATP synthase. ETC complexes organize into supramolecular structures called supercomplexes (ETC SCs). Protein factors regulating ETC SCs remain largely unknown despite their fundamental implications to mitochondrial respiratory function. Recent knock-out studies have delineated external ETC proteins HIGD1A and HIGD2A as assembly factors of ETC complexes III and IV, and their incorporation into SCs. In order to clarify the primary functions of HIGD1A and HIGD2A, as well as other previously uncharacterized HIG1 protein family members, stable overexpression (OE) models of each HIG1 protein were generated in HEK293t cells to preform comparative studies. We uncover a general dichotomy in the effects observed from HIGD2A vs. HIGD1A/1B/1C OE. Furthermore, we demonstrate that the previously unstudied protein family member HIGD1C is a negative regulator of complex IV SCs. A very limited number of protein factors specifically regulating the I1III2IV1 “respirasome” ETC SC have been identified. We propose a new framework where select complex I accessory subunits regulate respirasome assembly through protein-protein interactions between ETC complexes. Through specific point mutations to one such subunit, we generate a novel cell model with selective disassembly of the respirasome but otherwise functional individual ETC complexes. We demonstrate that respirasome disassembly limits respiration and modifies electron transfer pathways within the ETC. These findings to respirasome assembly and function may represent just a portion of higher order regulation that we are beginning to describe within eukaryotic metabolism.

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