Abstract
SummaryMammalian 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.
Highlights
Mammalian complex I can reversibly adopt catalytically active (A-) and catalytically deactive (D-) states
The ability of mammalian complex I to form these distinct states was foreshadowed in 1950, when a gradual loss in NADH oxidase activity was noted after incubation at 37C (Slater, 1950) that was prevented by NADH (Luzikov et al, 1970; Rossi et al, 1965)
Complex I inhibition by electrophiles, e.g., N-ethyl maleimide (NEM), was subsequently ascribed to modification of the ND3 subunit Cys39 residue, which appeared to be selectively exposed in the catalytically inactive D-state (Galkin et al, 2008). This led to a model of the catalytic A/D transition, in which Cys39 was occluded in the catalytic A-state, rendering complex I resistant to thiol reagents, but exposed to reaction with electrophiles in the D-state, that prevented reactivation of catalytic turnover by NADH (Figure 1)
Summary
Mammalian complex I can reversibly adopt catalytically active (A-) and catalytically deactive (D-) states. The ability of mammalian complex I to form these distinct states was foreshadowed in 1950, when a gradual loss in NADH oxidase activity was noted after incubation at 37C (Slater, 1950) that was prevented by NADH (Luzikov et al, 1970; Rossi et al, 1965). These observations were rationalized by demonstrating the transition between these states in submitochondrial particles (Kotlyar and Vinogradov, 1990). This led to a model of the catalytic A/D transition, in which Cys was occluded in the catalytic A-state, rendering complex I resistant to thiol reagents, but exposed to reaction with electrophiles in the D-state, that prevented reactivation of catalytic turnover by NADH (Figure 1)
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