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.
|Hiser, Carrie; Liu, Jian; Ferguson-Miller, Shelagh (2018) The K-path entrance in cytochrome c oxidase is defined by mutation of E101 and controlled by an adjacent ligand binding domain. Biochim Biophys Acta Bioenerg 1859:725-733|
|Liu, Jian; Hiser, Carrie; Ferguson-Miller, Shelagh (2017) Role of conformational change and K-path ligands in controlling cytochrome c oxidase activity. Biochem Soc Trans 45:1087-1095|
|Liu, Jian; Hiser, Carrie; Ferguson-Miller, Shelagh (2017) Correction: Role of conformational change and K-path ligands in controlling cytochrome c oxidase activity. Biochem Soc Trans 45:1345|
|Li, Fei; Liu, Jian; Liu, Nan et al. (2016) Translocator Protein 18 kDa (TSPO): An Old Protein with New Functions? Biochemistry 55:2821-31|
|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; 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; Garavito, R Michael et al. (2015) Evolving understanding of translocator protein 18 kDa (TSPO). Pharmacol Res 99:404-9|
|Li, Fei; Liu, Jian; Zheng, Yi et al. (2015) Response to Comment on ""Crystal structures of translocator protein (TSPO) and mutant mimic of a human polymorphism"". Science 350:519|
|Schwaighofer, Andreas; Ferguson-Miller, Shelagh; Naumann, Renate L C et al. (2014) Phase-sensitive detection in modulation excitation spectroscopy applied to potential induced electron transfer in cytochrome c oxidase. Appl Spectrosc 68:5-13|
|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|
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