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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026916-34
Application #
8448773
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Anderson, Vernon
Project Start
1979-07-01
Project End
2015-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
34
Fiscal Year
2013
Total Cost
$385,257
Indirect Cost
$124,638
Name
Michigan State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
193247145
City
East Lansing
State
MI
Country
United States
Zip Code
48824
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
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
Liu, Jian; Qin, Ling; Ferguson-Miller, Shelagh (2011) Crystallographic and online spectral evidence for role of conformational change and conserved water in cytochrome oxidase proton pump. Proc Natl Acad Sci U S A 108:1284-9
Zhang, Xi; Hiser, Carrie; Tamot, Banita et al. (2011) Combined genetic and metabolic manipulation of lipids in Rhodobacter sphaeroides reveals non-phospholipid substitutions in fully active cytochrome c oxidase. Biochemistry 50:3891-902
Zhang, Xi; Tamot, Banita; Hiser, Carrie et al. (2011) Cardiolipin deficiency in Rhodobacter sphaeroides alters the lipid profile of membranes and of crystallized cytochrome oxidase, but structure and function are maintained. Biochemistry 50:3879-90
Mills, Denise A; Xu, Shujuan; Geren, Lois et al. (2008) Proton-dependent electron transfer from CuA to heme a and altered EPR spectra in mutants close to heme a of cytochrome oxidase. Biochemistry 47:11499-509
Varanasi, Lakshman; Mills, Denise; Murphree, Anna et al. (2006) Altering conserved lipid binding sites in cytochrome c oxidase of Rhodobacter sphaeroides perturbs the interaction between subunits I and III and promotes suicide inactivation of the enzyme. Biochemistry 45:14896-907
Qin, Ling; Hiser, Carrie; Mulichak, Anne et al. (2006) Identification of conserved lipid/detergent-binding sites in a high-resolution structure of the membrane protein cytochrome c oxidase. Proc Natl Acad Sci U S A 103:16117-22
Mills, Denise A; Hosler, Jonathan P (2005) Slow proton transfer through the pathways for pumped protons in cytochrome c oxidase induces suicide inactivation of the enzyme. Biochemistry 44:4656-66
Branden, Gisela; Branden, Magnus; Schmidt, Bryan et al. (2005) The protonation state of a heme propionate controls electron transfer in cytochrome c oxidase. Biochemistry 44:10466-74

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