Reaction Mechanism of Single Subunit NADH-Ubiquinone Oxidoreductase (Ndi1) from Saccharomyces cerevisiae

Abstract

The flavoprotein rotenone-insensitive internal NADHubiquinone (UQ) oxidoreductase (Ndi1) is a member of the respiratory chain in Saccharomyces cerevisiae. We reported previously that bound UQ in Ndi1 plays a key role in preventing the generation of reactive oxygen species. Here, to elucidate this mechanism, we investigated biochemical properties of Ndi1 and its mutants in which highly conserved amino acid residues (presumably involved in NADH and/or UQ binding sites) were replaced. We found that wild-type Ndi1 formed a stable charge transfer (CT) complex (around 740 nm) with NADH, but not with NADPH, under anaerobic conditions. The intensity of the CT absorption band was significantly increased by the presence of bound UQ or externally added n-decylbenzoquinone. Interestingly, however, when Ndi1 was exposed to air, the CT band transiently reached the same maximum level regardless of the presence of UQ. This suggests that Ndi1 forms a ternary complex with NADH and UQ, but the role of UQ in withdrawing an electron can be substitutable with oxygen. Proteinase K digestion analysis showed that NADH (but not NADPH) binding induces conformational changes in Ndi1. The kinetic study of wild-type and mutant Ndi1 indicated that there is no overlap between NADH and UQ binding sites. Moreover, we found that the bound UQ can reversibly dissociate from Ndi1 and is thus replaceable with other quinones in the membrane. Taken together, unlike other NAD(P)H-UQ oxidoreductases, the Ndi1 reaction proceeds through a ternary complex (not a ping-pong) mechanism. The bound UQ keeps oxygen away from the reduced flavin. Alternative NADH dehydrogenases (NDH-2)4 catalyze electron transfer from NADH to quinone without energy transduction. They are commonly found in the respiratory chain of bacteria, fungi, and plant mitochondria but not in mammalian mitochondria (1, 2). The NDH-2 enzyme is generally composed of a single polypeptide and can be classified into three groups: A, B, and C. Group A has two adenine dinucleotide (ADP)-binding motifs and is involved in the non-covalent binding of NAD(P)H and FAD. Group B has the same two ADP-binding motifs and an additional conserved EF-hand. Group C has only one conserved ADP-binding motif where flavin is covalently bound (2). The yeast Ndi1 enzyme belongs to group A (2) and catalyzes NADH oxidation on the matrix side of the mitochondrial inner membrane (3) like the energy-transducing NADH dehydrogenase complex I (4).The physiological electron acceptor of NDH-2 is quinone, which is broadly classified into two types: naphthoquinone (menaquinone) and benzoquinone (ubiquinone (UQ)). Ndi1 utilizes the latter as an electron acceptor (2). Both quinones have a hydrophilic head group and hydrophobic isoprenoid side chains and ensure electron transfer between several membrane-bound respiratory enzymes. Ndi1 does not react with oxygen in the presence of UQ. In the absence of quinone, NADH-reduced Ndi1 reacts with oxygen and produces H 2 O 2 , although intrinsic NADH oxidase activity is extremely low (about 30...

The structure of the yeast NADH dehydrogenase (Ndi1) reveals overlapping binding sites for water- and lipid-soluble substrates

“…However, a recent examination of the reaction mechanism of Ndi1 by site-directed mutagenesis, proteinase K digestion, and kinetic analyses concluded that the reaction proceeds through a ternary complex (26). The results of the mutagenesis experiments reported in this study agree well with the present structural analysis, placing residues Pro-92 and Leu-93 in close proximity to the FAD cofactor (Fig.…”

“…S6). In addition, the observation of a single UQ2-binding site per monomer of Ndi1 is consistent with the most recent biochemical and kinetic analyses of the enzyme (26).…”

“…S2B); however, the existence of a ternary complex, where Ndi1 is associated with NADH and quinone simultaneously, is inconsistent with the current structural data, which show coincident binding sites for NAD + (but not NADH) and UQ2. The proteolytic digestion analyses reported by Yang et al (26) proposed a conformational change in the structure of Ndi1, induced by NADH (but not NAD + ), which is essential for the formation of a ternary complex. This analysis is not supported by the present structures, which show no molecular rearrangement on NAD + or UQ2 binding.…”

“…S2). Yang et al (26) identified residues Pro-92 and Leu-93 as being positioned close to the FAD-binding site. These results are entirely consistent with the structure of Ndi1 as described here, which places residues Pro-92 and Thr-91 on the siface of the FAD cofactor, with a hydrogen-bonding interaction between Thr-91 and the FAD.…”

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

“…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.…”