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

Abstract

Bioenergy is efficiently produced in the mitochondria by the respiratory system consisting of complexes I-V. In various organisms, complex I can be replaced by the alternative NADH-quinone oxidoreductase (NDH-2), which catalyzes the transfer of an electron from NADH via FAD to quinone, without proton pumping. The Ndi1 protein from Saccharomyces cerevisiae is a monotopic membrane protein, directed to the matrix. A number of studies have investigated the potential use of Ndi1 as a therapeutic agent against complex I disorders, and the NDH-2 enzymes have emerged as potential therapeutic targets for treatments against the causative agents of malaria and tuberculosis. Here we present the crystal structures of Ndi1 in its substrate-free, NAD + -and ubiquinone-(UQ2) complexed states. The structures reveal that Ndi1 is a peripheral membrane protein forming an intimate dimer, in which packing of the monomeric units within the dimer creates an amphiphilic membrane-anchor domain structure. Crucially, the structures of the Ndi1-NAD + and Ndi1-UQ2 complexes show overlapping binding sites for the NAD + and quinone substrates.alternative complex I | structural biology B ioenergy is efficiently produced in the mitochondria by the respiratory system consisting of complexes I-V. The first of these complexes, NDH-1 (complex I, proton pumping NADH-Q oxidoreductase) serves as the major entry point into the respiratory chain, for electrons derived from metabolic processes. In various organisms, complex I can be replaced by the alternative NADH-quinone oxidoreductase (NDH-2), which catalyzes the transfer of an electron from NADH via FAD to quinone, without proton pumping (1). The NDH-2 enzymes are found in bacteria and the mitochondria of plants and fungi, but crucially, not in mammalian mitochondria. Plant and fungal mitochondria possess two types of NDH-2: one is directed to the matrix and catalyzes NADH oxidation in the matrix (designated the internal NADH dehydrogenase or Ndi), and the other faces the intermembrane space and oxidizes NADH in the cytoplasmic space (designated the external NADH dehydrogenase or Nde).

Structures of the PutA peripheral membrane flavoenzyme reveal a dynamic substrate-channeling tunnel and the quinone-binding site

“…S5A). The arm is reminiscent of the hydrophobic ridge of yeast NADH dehydrogenase that is proposed to contact the membrane (23), and involvement of the α domain in membrane association is consistent with the observation of a detergent molecule bound to this region (Fig. 4E) and fluorescence quenching studies of E. coli PutA (31).…”

“…The quinone ring binds within 4 Å of the isoalloxazine, which is consistent with direct electron transfer and reminiscent of other flavoenzyme-quinone complexes (22)(23)(24), especially NAD(P)H/ quinone acceptor oxidoreductase (25,26). Although both MB and THFA bind at the si face of the isoalloxazine, the two ligands associate with different conformations of the active site and provoke different protein conformational changes (Fig.…”

“…This face of GsPutA presents adjacent regions of neutral and positive electrostatic potential (Fig. S5A), which is the expected pattern for a peripheral membrane association surface (23,(28)(29)(30). In contrast, the convex face (Fig.…”

Apoptosis-inducing Factor (AIF) and Its Family Member Protein, AMID, Are Rotenone-sensitive NADH:Ubiquinone Oxidoreductases (NDH-2)

“…These results prompted us to consider the possibility that the C-terminal polyhistidine tag might inhibit AIF association to the membrane. To explore this idea, we compared the x-ray crystal structures of mouse AIF (44) with that of Ndi1 from S. cerevisiae (45,46) and NDH-2 from Caldalkalibacillus thermarum (47). In both the Ndi1 and NDH-2 enzymes, the C-terminal regions are essential for membrane anchoring.…”

Regulatory role of the respiratory supercomplex factors in Saccharomyces cerevisiae

“…In mammalian mitochondria, the first component of the respiratory chain is the integral membranebound complex I (or type I NADH dehydrogenase), which receives electrons from NADH. In Saccharomyces cerevisiae, complex I is replaced by type II NADH dehydrogenases, which are peripheral dimeric membrane proteins (1,2). In both cases, the electrons are used for reduction of quinone to quinol, which diffuses within the membrane to donate electrons to complex III [ubiquinol-cytochrome c reductase (cyt.…”