The central theme of this program involves studies of the mechanisms of action of the drug-metabolizing enzymes cytochrome P450 and other heme proteins such as myoglobin and cytochrome c. We also investigate metalloporphyrin model compounds to elucidate how these enzymatic processes occur. The principal approaches involve kinetic and mechanistic studies of enzyme-substrate interactions, the synthesis and characterization of reactive iron porphyrin species as models of putative enzymic intermediates and to relate the interconversions of these species toward a molecular understanding of these proteins. Two additional applications of the model compounds have evolved in the course of this project. We have discovered that some iron porphyrins are highly bioactive in suppressing protein nitration by peroxynitrite. Other metalloporphyrins have emerged as effective biomimics of P450 action, alowing the facile production of useful quantities of drug metabolites, often a bottleneck in drug development. Cytochrome P450 is the central protein involved in drug detoxification and hormone metabolism while the related nitric oxide synthase is the source of the signal molecules nitric oxide and peroxynitrite. Synthetic metalloporphyrins can be employed as probes to intervene in these processes in diagnostic ways. Thus, these agents may prove to be significant tools for elaborating the biology of superoxide, peroxynitrite and NO. These same metalloporphyrins have shown impressive activity in animals suggesting their application as pharmaceutical agents in degenerative diseases such as diabetes, cardiomyopathy ang age-related disorders. Our effort seeks to provide a foundation of mechanistic and kinetic information that can be applied to in vitro models, cell culture studies and whole animal models of specific disease states such as ischemia-reperfusion, sepsis and autoimmune diseases. Experiments are aimed at determining what reactive intermediates are formed and what their biological targets are likely to be. The elaboration of these processes will facilitate the design of metal complexes for the catalytic decomposition of peroxynitrite and these other species, while studies of protein tyrosine nitration will elucidate how proteins are damaged under conditions of oxidative and nitrosative stress. Novel types of rapid kinetic analysis have been developed to study the reactivity observed in these processes. Binding of cytochrome c to synthetic and semi-synthetic phospholipid assemblies, which afford a system of intermediate complexity, are used to model and understand the larger scale events in the role of cytochrome c in triggering lipid oxidation and programmed cell death (apoptosis).

Public Health Relevance

Cytochrome P450 enzymes mediate phase 1 drug metabolism. Other heme proteins such as myoglobin and cytochrome c are involved in nitric oxide catabolism and the initiation of programed cell death (apoptosis). Our program aims to understand these processes. The outcome could lead to more efficatious drugs and new ways of controlling tissue damage that arise from oxidative and nitrosative stress.

Agency
National Institute of Health (NIH)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM036298-29
Application #
8633039
Study Section
No Study Section (in-house review) (NSS)
Program Officer
Okita, Richard T
Project Start
Project End
Budget Start
Budget End
Support Year
29
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Princeton University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Princeton
State
NJ
Country
United States
Zip Code
08543
Groves, John T (2014) Enzymatic C-H bond activation: Using push to get pull. Nat Chem 6:89-91
Liu, Wei; Huang, Xiongyi; Groves, John T (2013) Oxidative aliphatic C-H fluorination with manganese catalysts and fluoride ion. Nat Protoc 8:2348-54
Wang, Xiaoshi; Peter, Sebastian; Ullrich, Rene et al. (2013) Driving force for oxygen-atom transfer by heme-thiolate enzymes. Angew Chem Int Ed Engl 52:9238-41
Bergstrom, Chris L; Beales, Paul A; Lv, Yang et al. (2013) Cytochrome c causes pore formation in cardiolipin-containing membranes. Proc Natl Acad Sci U S A 110:6269-74
Cooper, Harriet L R; Groves, John T (2011) Molecular probes of the mechanism of cytochrome P450. Oxygen traps a substrate radical intermediate. Arch Biochem Biophys 507:111-8
Peter, Sebastian; Kinne, Matthias; Wang, Xiaoshi et al. (2011) Selective hydroxylation of alkanes by an extracellular fungal peroxygenase. FEBS J 278:3667-75
Beales, Paul A; Bergstrom, Chris L; Geerts, Nienke et al. (2011) Single vesicle observations of the cardiolipin-cytochrome C interaction: induction of membrane morphology changes. Langmuir 27:6107-15
Surmeli, N Basak; Litterman, Nadia K; Miller, Anne-Frances et al. (2010) Peroxynitrite mediates active site tyrosine nitration in manganese superoxide dismutase. Evidence of a role for the carbonate radical anion. J Am Chem Soc 132:17174-85
Su, Jia; Groves, John T (2010) Mechanisms of peroxynitrite interactions with heme proteins. Inorg Chem 49:6317-29
Su, Jia; Groves, John T (2009) Direct detection of the oxygen rebound intermediates, ferryl Mb and NO2, in the reaction of metmyoglobin with peroxynitrite. J Am Chem Soc 131:12979-88

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