The proton-translocating NADH-quinone oxidoreductase is one of the four enzyme complexes in the respiratory chain of most aerobic species from bacteria to human. The enzyme is collectively called complex I and its bacterial counterpart is specifically referred to as NDH-1. Complex I/NDH-1 is recognized to have the most intricate structure of the membrane-associated enzyme complexes. Low resolution EM analyses indicate that the enzyme has an L-shaped structure consisting of a membrane domain and a peripheral domain. Recently, high resolution 3D structure of the peripheral domain of NDH-1 became available. Complex I/NDH-1 contains one FMN and eight or nine FeS clusters as cofactors. These cofactors are located in the peripheral domain. This enzyme pumps protons and builds an electrochemical gradient across the membrane. Mammalian complex I is located in the innermitochondrial membrane and is composed of at least 45 different subunits with a total molecular mass of approximately 1 MDa. It is known that structural and functional defects of complex I are involved in many human mitochondrial diseases. Furthermore, sporadic Parkinson's disease is also related to complex I deficiencies. In contrast to the mitochondrial enzyme, NDH-1 has a simpler structure and is composed of 14 subunits. Because of its structural simplicity and availability of the 3D structure of the peripheral segment, NDH-1 serves as a useful model system for studying the structure and function of the mammalian enzyme. There is another type of NADH-quinone oxidoreductase named NDH-2 that is not found in mammals. NDH-2 is composed of a single polypeptide and does not pump protons. We have demonstrated that the yeast Ndi1, a member of NDH-2, can protect animals against complex I deficiencies. The overall goal of this grant application is to elucidate the structure and the mechanism of action of complex I/NDH-1 and Ndi1. The studies planned for this grant period are as follows: (1) Identification and characterization of the essential amino acid residues in the membrane domain subunits of NDH-1;(2) Clarification of functional roles of FeS clusters of NDH-1;(3) Identification of the Q-binding site and the inhibitor-binding site of NDH-1;(4) Clarification of the structure and the mechanism of action of the yeast Ndi1. Defects of mitochondrial NADH dehydrogenase cause many human diseases including sporadic Parkinson's disease. In order to develop therapies for diseases caused by deficiencies of this enzyme, it is a prerequisite to investigate why and how defects occur.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033712-25
Application #
7990400
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Anderson, Vernon
Project Start
1984-12-01
Project End
2012-11-30
Budget Start
2010-12-01
Budget End
2012-11-30
Support Year
25
Fiscal Year
2011
Total Cost
$611,807
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
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
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; 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
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
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
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
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
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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