Mitochondrial Complex II, also known as succinate dehydrogenase (SDH) is a membrane-bound heterotetramer (SDH-ABCD) whose four subunits are encoded by nuclear DNA. Complex II has a dual function in the cell by linking two essential energy-producing processes. As part of the TCA cycle the hydrophilic SDHAB domain oxidizes succinate to fumarate. This oxidation generates electrons that are transferred through the Fe-S clusters of the SDHB subunit to reduce ubiquinone within the SDHCD membrane domain to provide reducing equivalent to the electron transport chain needed for energy generation. As a key metabolic enzyme malfunction of Complex II is associated with debilitating neurodegenerative diseases and tumor formation. Although a significant amount of information is available on the structure and function of mature Complex II, there is gap in our understanding of how the enzyme assembles into a functional complex. In this project we will identify how various assembly factors work together to insert the flavin (FAD) and Fe-S redox centers into the SDHAB subunits and how this process is coordinated to assemble a functional Complex II.
Aim 1 : Our recent progress has provided insight into how assembly chaperones interact with the bacterial SdhA subunit to form the covalent FAD linkage needed for catalysis. We will expand on these findings to show how substrates enable protein domain movements to activate this assembly process and control catalysis in both bacterial SdhA and human SDHA subunits. This will be accomplished by x-ray crystallography, SAXS, and double electron electron resonance (DEER) spectroscopy of different intermediate assembly states.
Aim 2 : It is known that the Fe-S containing subunit requires specific assembly factors to incorporate its clusters. In this aim we investigate how the human SDHB (or bacterial SdhB) subunit incorporates its three unique Fe-S clusters localized into two separate protein domains. We also will determine whether the unique Complex II assembly chaperones assist with the insertion of the Fe-S clusters, or whether they stabilize the protein intermediates for interaction with the rest of the cellular Fe-S assembly machinery.
Aim 3 : Either malfunction of Complex II assembly or disassociation of the assembled complex during ischemia/reperfusion can contribute pathologies in mitochondrial function. In this aim we reveal mechanisms of how the SDHA and SDHB subunits form a functional subcomplex and aberrant activities associated with accumulation of the SDHAB subcomplex. A focus of this aim is how assembly factors and other proteins control the potentially deleterious reactive oxygen species that can be formed from the SDHAB subcomplex.

Public Health Relevance

Complex II (succinate dehydrogenase) is an essential metabolic enzyme involved in generation of energy for cells. In humans, a partial loss of activity of Complex II through mutation, misassembly, or complex disruption can lead to tumors, neurodegeneration, or cardiac dysfunction. The research in this proposal will determine the process of how Complex II assembles into a functional mitochondrial protein complex and characterize aberrant activity of misassesembled Complex II; this information will improve our understanding of human diseases associated with changes in Complex II function.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM061606-17
Application #
9685604
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Anderson, Vernon
Project Start
2001-07-01
Project End
2022-11-30
Budget Start
2019-01-04
Budget End
2019-11-30
Support Year
17
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Northern California Institute Research & Education
Department
Type
DUNS #
613338789
City
San Francisco
State
CA
Country
United States
Zip Code
94121
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; 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
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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