The importance of developing a better understanding of the function and regulation of cytochrome c oxidase has become increasingly apparent, given its decisive influence on mitochondrial metabolism and the central role of mitochondria in controlling cell life and death. Research supported by this grant has led us to a new perspective on this complex energy conserving machine, derived from a number of new high resolution structures of the enzyme from the mitochondrial model system, Rhodobacter sphaeroides. These reveal previously unobserved changes in conformation associated with altered redox state, and the presence of lipid and steroid binding sites conserved in bacteria and mammals. This proposal is aimed at determining the significance of the novel structural findings through further crystallographic efforts designed to obtain new and higher resolution crystal forms, and through studies of the effects of lipidic ligands on activity, stability and efficiency of oxidase.
The Specific Aims are: 1) to generate additional crystal forms of two and four subunit Rhodobacter oxidase, using molecular engineering strategies and robotic crystal screening;2) to create, characterize and crystallize mutants that facilitate the trapping of novel catalytic intermediates or that restrain flexibility, to look for new conformational states and test the importance of conformational change;3) to screen for alternative ligands of a steroid binding site, with potential physiological significance, or inhibitory or stabilizing effects. A major tool in these studies will be crystallography, but our ability to comprehensively analyze oxidase function and spectral features, including on-line crystal spectra, will be crucial to interpreting the structural findings. The expected outcome is a new level of understanding of the molecular mechanism of energy conversion in cytochrome oxidase, including the role of conformational change in gating and efficiency, and the regulatory effects of lipidic ligands. The long term goal is to better understand the involvement of cytochrome oxidase in metabolic disease states including cancer, obesity, diabetes and aging, through structure/function analysis and the discovery of compounds that are physiological effectors, crystallization aids, mechanistic probes, or precursors to drugs that can modulate oxidase activity.
Cytochrome c oxidase is a critical player in normal physiological function, consuming more than 90% of the oxygen we breathe and being directly involved in the production of most of the energy we use to support all life processes;the goal of this research program is to develop a better understanding of cytochrome oxidase function and regulation. The importance of this objective has become increasingly apparent, given the decisive influence of cytochrome oxidase on mitochondrial energy metabolism and the central role of mitochondria in controlling cell life and death. The long term goal is to better understand the involvement of cytochrome oxidase in metabolic disease states including cancer, obesity, diabetes and aging, through structure/ function analysis and the discovery of new compounds that are physiological effectors, crystallization aids, mechanistic probes, or precursors to drugs that can modulate oxidase activity and efficiency.
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|Li, Fei; Liu, Jian; Valls, Lance et al. (2015) Identification of a key cholesterol binding enhancement motif in translocator protein 18 kDa. Biochemistry 54:1441-3|
|Li, Fei; Liu, Jian; Zheng, Yi et al. (2015) Protein structure. Crystal structures of translocator protein (TSPO) and mutant mimic of a human polymorphism. Science 347:555-8|
|Li, Fei; Liu, Jian; Garavito, R Michael et al. (2015) Evolving understanding of translocator protein 18 kDa (TSPO). Pharmacol Res 99:404-9|
|Buhrow, Leann; Hiser, Carrie; Van Voorst, Jeffrey R et al. (2013) Computational prediction and in vitro analysis of potential physiological ligands of the bile acid binding site in cytochrome c oxidase. Biochemistry 52:6995-7006|
|Li, Fei; Xia, Yan; Meiler, Jens et al. (2013) Characterization and modeling of the oligomeric state and ligand binding behavior of purified translocator protein 18 kDa from Rhodobacter sphaeroides. Biochemistry 52:5884-99|
|Hiser, Carrie; Buhrow, Leann; Liu, Jian et al. (2013) A conserved amphipathic ligand binding region influences k-path-dependent activity of cytochrome C oxidase. Biochemistry 52:1385-96|
|Buhrow, Leann; Ferguson-Miller, Shelagh; Kuhn, Leslie A (2012) From static structure to living protein: computational analysis of cytochrome c oxidase main-chain flexibility. Biophys J 102:2158-66|
|Szundi, Istvan; Funatogawa, Chie; Cassano, Jennifer et al. (2012) Spectral identification of intermediates generated during the reaction of dioxygen with the wild-type and EQ(I-286) mutant of Rhodobacter sphaeroides cytochrome c oxidase. Biochemistry 51:9302-11|
|Ferguson-Miller, Shelagh; Hiser, Carrie; Liu, Jian (2012) Gating and regulation of the cytochrome c oxidase proton pump. Biochim Biophys Acta 1817:489-94|
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