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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Anderson, Vernon
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Northern California Institute Research & Education
San Francisco
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Starbird, C A; Tomasiak, Thomas M; Singh, Prashant K et al. (2018) New crystal forms of the integral membrane Escherichia coli quinol:fumarate reductase suggest that ligands control domain movement. J Struct Biol 202:100-104
Tso, Shih-Chia; Chen, Qiuyan; Vishnivetskiy, Sergey A et al. (2018) Using two-site binding models to analyze microscale thermophoresis data. Anal Biochem 540-541:64-75
Sharma, Pankaj; Maklashina, Elena; Cecchini, Gary et al. (2018) Crystal structure of an assembly intermediate of respiratory Complex II. Nat Commun 9:274
Maklashina, Elena; Rajagukguk, Sany; Iverson, T M et al. (2018) The unassembled flavoprotein subunits of human and bacterial complex II have impaired catalytic activity and generate only minor amounts of ROS. J Biol Chem 293:7754-7765
Starbird, C A; Maklashina, Elena; Sharma, Pankaj et al. (2017) Structural and biochemical analyses reveal insights into covalent flavinylation of the Escherichia coli Complex II homolog quinol:fumarate reductase. J Biol Chem 292:12921-12933
Maklashina, Elena; Rajagukguk, Sany; Starbird, Chrystal A et al. (2016) Binding of the Covalent Flavin Assembly Factor to the Flavoprotein Subunit of Complex II. J Biol Chem 291:2904-16
Cheng, Victor W T; Piragasam, Ramanaguru Siva; Rothery, Richard A et al. (2015) Redox state of flavin adenine dinucleotide drives substrate binding and product release in Escherichia coli succinate dehydrogenase. Biochemistry 54:1043-52
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
Birmingham, William R; Starbird, Chrystal A; Panosian, Timothy D et al. (2014) Bioretrosynthetic construction of a didanosine biosynthetic pathway. Nat Chem Biol 10:392-9

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