Abstract
Type II NADH:quinone oxidoreductase (NDH-2) is central to the respiratory chains of many organisms. It is not present in mammals so may be exploited as an antimicrobial drug target or used as a substitute for dysfunctional respiratory complex I in neuromuscular disorders. NDH-2 is a single-subunit monotopic membrane protein with just a flavin cofactor, yet no consensus exists on its mechanism. Here, we use steady-state and pre-steady-state kinetics combined with mutagenesis and structural studies to determine the mechanism of NDH-2 from Caldalkalibacillus thermarum. We show that the two substrate reactions occur independently, at different sites, and regardless of the occupancy of the partner site. We conclude that the reaction pathway is determined stochastically, by the substrate/product concentrations and dissociation constants, and can follow either a ping-pong or ternary mechanism. This mechanistic versatility provides a unified explanation for all extant data and a new foundation for the development of therapeutic strategies.
Highlights
Type-II NADH:quinone oxidoreductase (NADH dehydrogenase-2, NDH-2) is a membrane-bound dehydrogenase that oxidizes NADH and reduces quinones and is a central feature of the respiratory chain in many diverse species, but not in animals[1]
In a classical ternary complex mechanism, both substrates are bound together and both products released simultaneously (Fig. 1B), whereas in NDH-2 formation of a stable CTC suggests that NADH oxidation has already occurred to some extent, before the quinone reacts
The ternary complex proposed to be formed during NDH-2 catalysis is atypical because the ternary complex has the character of a substrate-product complex (Fig. 1C)
Summary
Spectra of NDH-2 define the oxidation and nucleotide-binding states. Panel A shows that addition of NADH to the oxidized enzyme both reduces the flavin (most evident at 350 to 500 nm) and forms a charge-transfer complex (CTC, evident above 550 nm with a maximum at 660 nm)[15,21]. The data provide an upper limit for KD of 2 μM — considerably lower than the values of 11.4 and 20.3 μM determined for S. aureus NDH-2 by titrating NADH and NAD+ onto the protein and monitoring the fluorescence from two nearby tryptophans[15].
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