ND3 Cys39 in complex I is exposed during mitochondrial respiration
Nils Burger, Andrew M. James, John F. Mulvey, Kurt Hoogewijs, Shujing Ding, Ian M. Fearnley, Marta Loureiro, Abigail A.I. Norman, Sabine Arndt, Amin Mottahedin, Olga Sauchanka, Richard C. Hartley, Thomas Krieg, Michael P. Murphy
Abstract
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.