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Mammalian complex I can adopt catalytically active (A-) or deactive (D-) states. A defining feature of the reversible transition between these two defined states is thought to be exposure of the ND3 subunit Cys39 residue in the D-state and its occlusion in the A-state. As the catalytic A/D transition is important in health and disease, we set out to quantify it by measuring Cys39 exposure using isotopic labeling and mass spectrometry, in parallel with complex I NADH/CoQ oxidoreductase activity. To our surprise, we found significant Cys39 exposure during NADH/CoQ oxidoreductase activity. Furthermore, this activity was unaffected if Cys39 alkylation occurred during complex I-linked respiration. In contrast, alkylation of catalytically inactive complex I irreversibly blocked the reactivation of NADH/CoQ oxidoreductase activity by NADH. Thus, Cys39 of ND3 is exposed in complex I during mitochondrial respiration, with significant implications for our understanding of the A/D transition and the mechanism of complex I.

Original publication

DOI

10.1016/j.chembiol.2021.10.010

Type

Journal article

Journal

Cell Chem Biol

Publication Date

21/04/2022

Volume

29

Pages

636 - 649.e14

Keywords

Cys39, NADH:ubiquinone oxidoreductase, active/deactive transition, complex I, ischemia-reperfusion (IR) injury, mitochondria, redox regulation, reverse electron transport (RET), Animals, Catalysis, Electron Transport Complex I, Mammals, Mitochondria, NAD, Respiration