This research program describes the structure/function of the membrane-bound respiratory Complex II (succinate:ubiquinone oxidoreductase/succinate dehydrogenase) and its bacterial homologues. The long-term objectives of this research program are to describe mechanisms of electron transfer through the enzyme to/from flavin to quinones. Complex II has a number of redox centers involved in the electron transfer process. These include a covalently-bound FAD cofactor, three distinct iron- sulfur clusters, a membrane-bound quinone, and a b heme cofactor. With the exception of the b heme prosthetic group all of these redox centers are known as essential components of the electron transfer pathway. In mitochondria Complex II is an essential component of both the citric acid cycle and the membrane-bound electron transport chain. Specific mutations in Complex II contribute to disease including, tumor formation, neurodegeneration, cardiac dysfunction, and premature aging. The majority of these mutations structurally map to the quinone-binding domain of Complex II. The molecular mechanisms of how these defects contribute to disease are still not understood. The studies described in this application have three general aims. The first is designed to describe the essential components of the quinone-binding site of Complex II and how the architecture of this site influences catalytic activity. Complex II is an excellent model for these studies since it has the ability to interact with both ubi- and napthoquinones. Thus, using a series of site-directed mutants and kinetic assays, Fourier transform infrared and pulsed EPR spectroscopy, and x-ray crystallography the necessary components for the proper functioning of the quinone-binding site will be defined. Second, by using the technique of pulse radiolysis, in conjunction with other methods, we will determine rate constants for electron transfer between specific pairs of redox-active centers in both wild-type and mutant forms of Complex II. It is hypothesized that altered kinetics of electron transfer contribute to Complex II dysfunction, which in turn leads to a cascade of metabolic events leading to disease. Thus, the role of the various redox centers, including the b heme in electron transfer reactions will be defined.
The final aim i s to characterize conformational changes of Complex II upon interaction with other proteins. Recent findings show that the Complex II homologue quinol:fumarate oxidoreductase (QFR, fumarate reductase) interacts with FliG, a component of the bacterial flagellar switch complex. This interaction is important for controlling the direction of flagellar rotation and assembly. These studies will be accomplished by mutagenesis and kinetic assays of the enzyme, structural analysis of the QFR:FliG complex, and site-directed spin labeling EPR spectroscopy.

Public Health Relevance

Complex II (succinate:ubiquinone oxidoreductase) is an essential metabolic component involved in mitochondrial metabolism. When Complex II does not function properly, this can lead to neurodegeneration, heart disease, and tumor formation. The studies described in this application are designed to understand how Complex II mutations (known to cause disease) affect the function of Complex II. These studies will also describe how Complex II interacts with other protein components in the cell using bacterial models for their mitochondrial counterparts. This may be important to show how protein complexes communicate with one another.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061606-12
Application #
8307543
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Anderson, Vernon
Project Start
2001-07-01
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2014-07-31
Support Year
12
Fiscal Year
2012
Total Cost
$336,123
Indirect Cost
$110,700
Name
Northern California Institute Research & Education
Department
Type
DUNS #
613338789
City
San Francisco
State
CA
Country
United States
Zip Code
94121
Melin, Frederic; Noor, Mohamed R; Pardieu, Elodie et al. (2014) Investigating the thermostability of succinate: quinone oxidoreductase enzymes by direct electrochemistry at SWNTs-modified electrodes and FTIR spectroscopy. Chemphyschem 15:3572-9
Anderson, Robert F; Shinde, Sujata S; Hille, Russ et al. (2014) Electron-transfer pathways in the heme and quinone-binding domain of complex II (succinate dehydrogenase). Biochemistry 53:1637-46
Maklashina, Elena; Cecchini, Gary; Dikanov, Sergei A (2013) Defining a direction: electron transfer and catalysis in Escherichia coli complex II enzymes. Biochim Biophys Acta 1827:668-78
Cheng, Victor W T; Tran, Quang M; Boroumand, Nasim et al. (2013) A conserved lysine residue controls iron-sulfur cluster redox chemistry in Escherichia coli fumarate reductase. Biochim Biophys Acta 1827:1141-7
Singh, Prashant K; Sarwar, Maruf; Maklashina, Elena et al. (2013) Plasticity of the quinone-binding site of the complex II homolog quinol:fumarate reductase. J Biol Chem 288:24293-301
Zarbiv, Gabriel; Li, Hui; Wolf, Amnon et al. (2012) Energy complexes are apparently associated with the switch-motor complex of bacterial flagella. J Mol Biol 416:192-207
Lovett, David H; Mahimkar, Rajeev; Raffai, Robert L et al. (2012) A novel intracellular isoform of matrix metalloproteinase-2 induced by oxidative stress activates innate immunity. PLoS One 7:e34177
Kareyeva, Alexandra V; Grivennikova, Vera G; Cecchini, Gary et al. (2011) Molecular identification of the enzyme responsible for the mitochondrial NADH-supported ammonium-dependent hydrogen peroxide production. FEBS Lett 585:385-9
Ruprecht, Jonathan; Iwata, So; Rothery, Richard A et al. (2011) Perturbation of the quinone-binding site of complex II alters the electronic properties of the proximal [3Fe-4S] iron-sulfur cluster. J Biol Chem 286:12756-65
Pyburn, Tasia M; Bensing, Barbara A; Xiong, Yan Q et al. (2011) A structural model for binding of the serine-rich repeat adhesin GspB to host carbohydrate receptors. PLoS Pathog 7:e1002112

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