This proposal seeks support for investigations into the generation, characterization and, most importantly, reactivity of the 4 least well-understood cytochrome P450 (and related enzyme) transient intermediates - those involving oxygen: oxy-ferrous, peroxo-ferric, hydroperoxo-ferric, and oxo-iron(IV) [compound I, Cpd I, oxo-iron(IV)porph+]. 2 similar oxo species, compounds II and ES, will also be examined.
3 specific aims will be pursued. First (1), rapid kinetics methods will explore the role of hydrogen bonding in formation of a newly-observed """"""""perturbed"""""""" oxy catalytic species by use of mutants with altered proximal and distal hydrogen bonding properties. Parallel studies of oxy NO synthase will use modified tetrahydrobiopterins to probe electron transfer. Second (2), the mechanism whereby peroxo and hydroperoxo P450 may serve as alternate oxidants will be tested using T252A P450-CAM, a mutant that forms those species but almost none of the primary oxidant, Cpd I. One-electron cryoreduction of oxy P450-CAM provides another way to study the peroxo and hydroperoxo states; solvent and substrate isotope effects on the annealing of the hydroperoxo state will reveal important mechanistic information about its reactivity.
Both Aim 2 approaches will further test the """"""""two oxidant"""""""" hypothesis of P450 reactivity. The third goal (3) is to characterize the properties and reactivities of transient oxo Cpd I (and related) intermediates. Having optimized conditions for P450-CAM Cpd I formation, we will use rapid freeze-quench methods to characterize it spectroscopically. This will establish its electronic properties, and help explain its reactivity. Double-mix stopped-flow experiments will examine Cpd I reactivity and clarify key mechanistic aspects of O atom transfer by peroxidases and P450. With P450-CAM, this will include the first direct reactions of Cpd I with substrates and determination of the intermolecular isotope effect for hydroxylation - a crucial test of the well-accepted """"""""oxygen rebound"""""""" mechanism of hydroxylation. The knowledge derived from this work will lead to a more complete understanding of how heme enzymes activate peroxide and dioxygen with important medical implications for human health and disease, especially as described below for P450,. With over 3700 genes, P450 cytochromes are among the most essential and ubiquitous enzymes known. In human health, 57 P450s are responsible for countless critical transformations in steroid, vitamin D, eicosanoid, as well as drug metabolism. In human disease, P450-aromatase is a target for breast cancer chemotherapy owing to its vital role in estrogen hormone biosynthesis and several P450s have been shown to be activators of procarcinogens such as polycyclic aromatic hydrocarbons and nitrosamines. Progress in comprehending the P450 mechanism will promote medical advances to address these health issues. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM026730-19A2
Application #
7144188
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Ikeda, Richard A
Project Start
1979-07-01
Project End
2010-06-30
Budget Start
2006-07-01
Budget End
2007-06-30
Support Year
19
Fiscal Year
2006
Total Cost
$275,255
Indirect Cost
Name
University of South Carolina at Columbia
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
041387846
City
Columbia
State
SC
Country
United States
Zip Code
29208
Modi, Anuja R; Dawson, John H (2015) Oxidizing intermediates in P450 catalysis: a case for multiple oxidants. Adv Exp Med Biol 851:63-81
Smith, Aaron D; Modi, Anuja R; Sun, Shengfang et al. (2015) Spectroscopic Determination of Distinct Heme Ligands in Outer-Membrane Receptors PhuR and HasR of Pseudomonas aeruginosa. Biochemistry 54:2601-12
Beltrán, Jesús; Kloss, Brian; Hosler, Jonathan P et al. (2015) Control of carotenoid biosynthesis through a heme-based cis-trans isomerase. Nat Chem Biol 11:598-605
Draganova, Elizabeth B; Akbas, Neval; Adrian, Seth A et al. (2015) Heme Binding by Corynebacterium diphtheriae HmuT: Function and Heme Environment. Biochemistry 54:6598-609
Zhong, Fangfang; Lisi, George P; Collins, Daniel P et al. (2014) Redox-dependent stability, protonation, and reactivity of cysteine-bound heme proteins. Proc Natl Acad Sci U S A 111:E306-15
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
Davydov, Roman; Dawson, John H; Perera, Roshan et al. (2013) The use of deuterated camphor as a substrate in (1)H ENDOR studies of hydroxylation by cryoreduced oxy P450cam provides new evidence of the involvement of compound I. Biochemistry 52:667-71
Molitor, Bastian; Stassen, Marc; Modi, Anuja et al. (2013) A heme-based redox sensor in the methanogenic archaeon Methanosarcina acetivorans. J Biol Chem 288:18458-72
Sun, Shengfang; Sono, Masanori; Dawson, John H (2013) Mono- and bis-phosphine-ligated H93G myoglobin: spectral models for ferrous-phosphine and ferrous-CO cytochrome P450. J Inorg Biochem 127:238-45
Owens, Cedric P; Du, Jing; Dawson, John H et al. (2012) Characterization of heme ligation properties of Rv0203, a secreted heme binding protein involved in Mycobacterium tuberculosis heme uptake. Biochemistry 51:1518-31

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