Mitochondrial complex I (NADH-ubiquinone reductase) bears coupling site I, and is composed of lesser than 25 unlike polypeptides, FMN and 8 iron-sulfur (FeS) clusters. Four FeS clusters are rapidly reduced by NADH, one has an Em lesser than -400 mV, and three are EPR silent. The polypeptides containing FMN and FeS clusters are encased within lesser than 17 hydrophobic polypeptides. The complexity of mammalian complex I has hampered progress in study of many aspects of its structure and function. The respiratory chain of aerobically grown Paracoccus denitrificans contains coupling site I, is inhibited by rotenone, and exhibits EPR signals similar to those of mitochondrial complex I. The cytochrome oxidase of this organism is also similar to the mitochondrial enzyme in terms of electron carriers (cytochromes aa3, 2Cu). However, the P. denitrificans oxidase is composed of only 2 polypeptides, while the mammalian enzyme contains 7 major and possibly 3 to 5 minor polypeptides. The purpose of this project is to isolate complex I from P. denitrificans and study its structure; polypeptide, flavin and FeS composition; and mechanisms of electron transfer and H+ translocation. Specific plans are: (a) Isolation from P. denitrificans of a rotenone-sensitive NADH-Q reductase; (b) analysis of the complex for number of polypeptides, and concentrations of flavin (also whether FMN or FAD); Fe, S2-, Q, phospholipids; (c) EPR studies of the FeS clusters and their Em; (d) resolution of the complex by chaotropes for possible isolation of flavoprotein and FeS protein fragments, and their analysis as in b to d; (e) studies with surface-labeling reagents to identify accessible polypeptides; (f) production of antibodies to the complex and resolved fragments, and their use for functional, topological and membrane orientation studies; (g) reconstitution of chaotrope-resolved fragments to understand electron transfer sequence and inhibition sites of rotenone, etc.; (h) use of protein residue modifiers for specific inhibition and possible discrimination between electron transfer and H+ translocation, and identification of components involved in H+ translocation; (i) isolation (directly or by immunoprecipitation) of the enzyme from rotenone-insensitive mutants or cells grown under sulfate-limited conditions. These membranes lack coupling site I and EPR signal due to FeS cluster 2. Comparative structural-functional studies with normal complex I should shed light on the mechanisms of H+ translocation at site I.

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National Institute of General Medical Sciences (NIGMS)
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Physical Biochemistry Study Section (PB)
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La Jolla Institute
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Sinha, Prem Kumar; Castro-Guerrero, Norma; Patki, Gaurav et al. (2015) Conserved amino acid residues of the NuoD segment important for structure and function of Escherichia coli NDH-1 (complex I). Biochemistry 54:753-64
Sato, Motoaki; Torres-Bacete, Jesus; Sinha, Prem Kumar et al. (2014) Essential regions in the membrane domain of bacterial complex I (NDH-1): the machinery for proton translocation. J Bioenerg Biomembr 46:279-87
Barker, Clive S; Meshcheryakova, Irina V; Sasaki, Toshio et al. (2014) Randomly selected suppressor mutations in genes for NADH?:?quinone oxidoreductase-1, which rescue motility of a Salmonella ubiquinone-biosynthesis mutant strain. Microbiology 160:1075-86
Sato, Motoaki; Sinha, Prem Kumar; Torres-Bacete, Jesus et al. (2013) Energy transducing roles of antiporter-like subunits in Escherichia coli NDH-1 with main focus on subunit NuoN (ND2). J Biol Chem 288:24705-16
Torres-Bacete, Jesus; Sinha, Prem Kumar; Sato, Motoaki et al. (2012) Roles of subunit NuoK (ND4L) in the energy-transducing mechanism of Escherichia coli NDH-1 (NADH:quinone oxidoreductase). J Biol Chem 287:42763-72
Iwata, Momi; Lee, Yang; Yamashita, Tetsuo et al. (2012) The structure of the yeast NADH dehydrogenase (Ndi1) reveals overlapping binding sites for water- and lipid-soluble substrates. Proc Natl Acad Sci U S A 109:15247-52
Sinha, Prem Kumar; Nakamaru-Ogiso, Eiko; Torres-Bacete, Jesus et al. (2012) Electron transfer in subunit NuoI (TYKY) of Escherichia coli NADH:quinone oxidoreductase (NDH-1). J Biol Chem 287:17363-73
Yang, Yu; Yamashita, Tetsuo; Nakamaru-Ogiso, Eiko et al. (2011) Reaction mechanism of single subunit NADH-ubiquinone oxidoreductase (Ndi1) from Saccharomyces cerevisiae: evidence for a ternary complex mechanism. J Biol Chem 286:9287-97
Torres-Bacete, Jesus; Sinha, Prem Kumar; Matsuno-Yagi, Akemi et al. (2011) Structural contribution of C-terminal segments of NuoL (ND5) and NuoM (ND4) subunits of complex I from Escherichia coli. J Biol Chem 286:34007-14
Murai, Masatoshi; Yamashita, Tetsuo; Senoh, Mai et al. (2010) Characterization of the ubiquinone binding site in the alternative NADH-quinone oxidoreductase of Saccharomyces cerevisiae by photoaffinity labeling. Biochemistry 49:2973-80

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