Through the research outlined in this competitive renewal, we seek a precise understanding of the mechanisms of biological oxidations through knowledge of the detailed chemistry and physical operation of oxygenase and oxidase catalysis. Proposed for investigation are the heme protein reductases, termed cytochromes P450, which play central and crucial roles in mammalian metabolism and human health. Understanding the functioningof the cytochrome P450 systems is critical to defining the control points of drug metabolism, pro-carcinogen processing, and the regulation and method for hormonal control of development: and gene regulation. Our approach is problem rather than technique or system focused. We choose amongst the 800+ known P450 sequences those cytochrome systems where there is substantial structural and functional background information. One such system is P450cam, from Pseudomonas putida, which catalyzes the regiospecific hydroxylation of camphor and with which we have been working since the inception of this grant. Other structurally defined P450 systems to be employed include P450(BM3) and P450eryF for which we have the genetic basis in hand to utilize the powerful tools of recombinant DNA technology to express a variety of variant proteins. We have focused our next funding period on four highly specific aims. First, to obtain further high resolution structural information fromx-- ray crystallography and physical - chemical definition of the intermediate states of iron and oxygen involved in the P450 catalytic cycle. Second, to further define and elucidate the mechanisms of acid base catalysis and proton delivery that are responsible for the unique chemistry displayed by the P450 cytochromes. Third, to understand the controlling features of the branch points in the catalytic cycle where the path is open to productive commitments for catalysis as well as various abortive autoxidative processes that leak reducing equivalents into the cell and form potentially toxic reduced dioxygen species. Finally, we seek to understand the role of amino acid residues proximal to the heme plane in control of redox movement in protein-protein complexes and the chemical structure/activities of intermediate states. Through these interdisciplinary efforts we hope to shed light on some of the most important problems of modern molecular biochemistry, providing insight into the inner workings of these processes and aiding therapeutic prescription and understanding of disease states through detailed mechanistic knowledge.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM031756-27
Application #
7472440
Study Section
Special Emphasis Panel (NSS)
Program Officer
Anderson, Vernon
Project Start
1982-08-01
Project End
2009-07-31
Budget Start
2008-08-01
Budget End
2009-07-31
Support Year
27
Fiscal Year
2008
Total Cost
$357,780
Indirect Cost
Name
University of Illinois Urbana-Champaign
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
041544081
City
Champaign
State
IL
Country
United States
Zip Code
61820
Denisov, Ilia G; Sligar, Stephen G (2017) Nanodiscs in Membrane Biochemistry and Biophysics. Chem Rev 117:4669-4713
Mak, Piotr J; Luthra, Abhinav; Sligar, Stephen G et al. (2014) Resonance Raman spectroscopy of the oxygenated intermediates of human CYP19A1 implicates a compound i intermediate in the final lyase step. J Am Chem Soc 136:4825-8
Khatri, Yogan; Gregory, Michael C; Grinkova, Yelena V et al. (2014) Active site proton delivery and the lyase activity of human CYP17A1. Biochem Biophys Res Commun 443:179-84
Khatri, Yogan; Luthra, Abhinav; Duggal, Ruchia et al. (2014) Kinetic solvent isotope effect in steady-state turnover by CYP19A1 suggests involvement of Compound 1 for both hydroxylation and aromatization steps. FEBS Lett 588:3117-22
Mak, Piotr J; Gregory, Michael C; Sligar, Stephen G et al. (2014) Resonance Raman spectroscopy reveals that substrate structure selectively impacts the heme-bound diatomic ligands of CYP17. Biochemistry 53:90-100
Gregory, Michael C; Denisov, Ilia G; Grinkova, Yelena V et al. (2013) Kinetic solvent isotope effect in human P450 CYP17A1-mediated androgen formation: evidence for a reactive peroxoanion intermediate. J Am Chem Soc 135:16245-7
Sun, Yuhan; Zeng, Weiqiao; Benabbas, Abdelkrim et al. (2013) Investigations of heme ligation and ligand switching in cytochromes p450 and p420. Biochemistry 52:5941-51
Numata, Mari; Grinkova, Yelena V; Mitchell, James R et al. (2013) Nanodiscs as a therapeutic delivery agent: inhibition of respiratory syncytial virus infection in the lung. Int J Nanomedicine 8:1417-27
Schuler, Mary A; Denisov, Ilia G; Sligar, Stephen G (2013) Nanodiscs as a new tool to examine lipid-protein interactions. Methods Mol Biol 974:415-33
Luthra, A; Gregory, M; Grinkova, Y V et al. (2013) Nanodiscs in the studies of membrane-bound cytochrome P450 enzymes. Methods Mol Biol 987:115-27

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