Type‐II NADH:quinone oxidoreductase from Staphylococcus aureus has two distinct binding sites and is rate limited by quinone reduction

Abstract

SummaryA prerequisite for any rational drug design strategy is understanding the mode of protein-ligand interaction. This motivated us to explore protein-substrate interaction in Type-II NADH:quinone oxidoreductase (NDH-2) from Staphylococcus aureus, a worldwide problem in clinical medicine due to its multiple drug resistant forms. NDHs-2 are involved in respiratory chains and recognized as suitable targets for novel antimicrobial therapies, as these are the only enzymes with NADH:quinone oxidoreductase activity expressed in many pathogenic organisms.We obtained crystal and solution structures of NDH-2 from S. aureus, showing that it is a dimer in solution. We report fast kinetic analyses of the protein and detected a charge-transfer complex formed between NAD + and the reduced flavin, which is dissociated by the quinone. We observed that the quinone reduction is the rate limiting step and also the only half-reaction affected by the presence of HQNO, an inhibitor. We analyzed protein-substrate interactions by fluorescence and STD-NMR spectroscopies, which indicate that NADH and the quinone bind to different sites. In summary, our combined results show the presence of distinct binding sites for the two substrates, identified quinone reduction as the rate limiting step and indicate the establishment of a NAD + -protein complex, which is released by the quinone.

The mechanism of catalysis by type-II NADH:quinone oxidoreductases

“…Because the reduction potential of the flavin (ca. −0.22 V15) is considerably higher than that of NADH (−0.32 V) the CTC is probably best described as NAD + bound to reduced flavin. The CTC is stable, suggesting that NAD + does not dissociate from the reduced flavin following hydride transfer, and panel B shows the CTC is also formed by addition of NAD + to the pre-reduced flavin.…”

“…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)1521. Because the reduction potential of the flavin (ca.…”

“…Three NDH-2 homologues have been characterized structurally, from Saccharomyces cerevisiae 1213, Caldalkalibacillus thermarum 14, and Staphylococcus aureus 15. All of them are homodimers, with each monomer containing two Rossmann folds that form nucleotide-binding sites for a noncovalently bound flavin adenine dinucleotide (FAD) and the NADH substrate.…”

“…1A). Alternatively, others have observed charge transfer complexes (CTCs, complexes in an intermediate state between NADH/oxidized flavin and NAD + /reduced flavin)1521 and suggested that the CTC is the species oxidized by quinone15 in a step in which both substrates are bound to the enzyme at the same time. In a classical ternary complex mechanism, both substrates are bound together and both products released simultaneously (Fig.…”

Role of Type 2 NAD(P)H Dehydrogenase NdbC in Redox Regulation of Carbon Allocation in Synechocystis

“…The primary structure of NDH-2 commonly includes two GXGXXG motifs within the b-sheet-a-helix-b-sheet domains (Rossman fold), one for binding NAD(P)H and another for FAD or FMN (Wierenga et al, 1986). Although the crystal structures of NDH-2 from Saccharomyces cerevisiae (Ndi1; Feng et al, 2012) and the bacterial type NDH-2 from Caldalkalibacillus thermarum (Heikal et al, 2014) as well as from Staphylococcus aureus (Sena et al, 2015) have been solved, the actual mechanism of NADH:quinone oxidoreduction still remains unclear despite recently presented models (Marreiros et al, 2017). It is generally agreed that, in organisms where NADH oxidation is carried out solely by NDH-2s, the main function of these enzymes is to perform the respiratory chainlinked NADH turnover and the following donation of electrons to the protein complexes that produce the H + gradient powering ATP production (for review, see Melo et al, 2004).…”