Molecular Probes of the Mechanisms of Cytocrhome P450

Summary

Principal Investigator: JOHN TAYLOR GROVES
Abstract: 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).
Funding Period: 1985-12-01 - 2017-03-31
more information: NIH RePORT

Top Publications

  1. pmc A highly reactive p450 model compound I
    Seth R Bell
    Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA
    J Am Chem Soc 131:9640-1. 2009
  2. pmc Oxidative aliphatic C-H fluorination with manganese catalysts and fluoride ion
    Wei Liu
    Department of Chemistry, Princeton University, Princeton, New Jersey, USA
    Nat Protoc 8:2348-54. 2013
  3. pmc Driving force for oxygen-atom transfer by heme-thiolate enzymes
    Xiaoshi Wang
    Department of Chemistry, Princeton University, Princeton, NJ 08544 USA
    Angew Chem Int Ed Engl 52:9238-41. 2013
  4. pmc Cytochrome c causes pore formation in cardiolipin-containing membranes
    Chris L Bergstrom
    Department of Chemistry, Princeton University, Princeton, NJ 08544, USA
    Proc Natl Acad Sci U S A 110:6269-74. 2013
  5. pmc Detection and kinetic characterization of a highly reactive heme-thiolate peroxygenase compound I
    Xiaoshi Wang
    Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States
    J Am Chem Soc 134:12897-900. 2012
  6. pmc Catalytic peroxynitrite decomposition improves reperfusion injury after heart transplantation
    Gabor Szabo
    Department of Cardiac Surgery, University of Heidelberg, Heidelberg, Germany
    J Thorac Cardiovasc Surg 143:1443-9. 2012
  7. pmc Selective hydroxylation of alkanes by an extracellular fungal peroxygenase
    Sebastian Peter
    Department of Bio and Environmental Sciences, International Graduate School of Zittau, Zittau, Germany
    FEBS J 278:3667-75. 2011
  8. pmc Single vesicle observations of the cardiolipin-cytochrome C interaction: induction of membrane morphology changes
    Paul A Beales
    Department of Chemical and Environmental Engineering, Yale University, New Haven, Connecticut 06511, United States
    Langmuir 27:6107-15. 2011
  9. pmc Peroxynitrite mediates active site tyrosine nitration in manganese superoxide dismutase. Evidence of a role for the carbonate radical anion
    N Basak Surmeli
    Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA
    J Am Chem Soc 132:17174-85. 2010
  10. pmc Molecular probes of the mechanism of cytochrome P450. Oxygen traps a substrate radical intermediate
    Harriet L R Cooper
    Department of Chemistry, Princeton University, Princeton, NJ 08544, USA
    Arch Biochem Biophys 507:111-8. 2011

Research Grants

  1. Structural bases of the functions of RNA-protein machines
    THOMAS ARTHUR STEITZ; Fiscal Year: 2013
  2. DEGENERATIVE AND DEMENTING DISEASES OF AGING
    Stanley B Prusiner; Fiscal Year: 2013
  3. Reactivity of Manganese and Iron Metalloenzyme Models
    David P Goldberg; Fiscal Year: 2013
  4. Peroxiredoxin 6 as an Anti-oxidant Enzyme
    Aron B Fisher; Fiscal Year: 2013
  5. BIOINORGANIC COPPER COORDINATION CHEMISTRY
    Kenneth D Karlin; Fiscal Year: 2013
  6. Radical Intermediates of Nitric Oxide Synthase &Myocardial Ischemia Reperfusion
    Ah Lim Tsai; Fiscal Year: 2013
  7. HORMONAL REGULATION OF BLOOD PRESSURE
    Michal Laniado Schwartzman; Fiscal Year: 2013
  8. IPF Fibroblast Phenotype
    Craig A Henke; Fiscal Year: 2013
  9. Semi-volatile PCBs: Sources, Exposures, Toxicities
    Larry W Robertson; Fiscal Year: 2013
  10. Electron Transfer in Iron and Copper Proteins
    Jay R Winkler; Fiscal Year: 2013

Detail Information

Publications13

  1. pmc A highly reactive p450 model compound I
    Seth R Bell
    Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA
    J Am Chem Soc 131:9640-1. 2009
    ..More generally, subtle charge modulation at the active site may result in high reactivity of a cytochrome P450 compound I...
  2. pmc Oxidative aliphatic C-H fluorination with manganese catalysts and fluoride ion
    Wei Liu
    Department of Chemistry, Princeton University, Princeton, New Jersey, USA
    Nat Protoc 8:2348-54. 2013
    ..The procedures are performed in a typical fume hood using ordinary laboratory glassware. No special precautions to rigorously exclude water are required...
  3. pmc Driving force for oxygen-atom transfer by heme-thiolate enzymes
    Xiaoshi Wang
    Department of Chemistry, Princeton University, Princeton, NJ 08544 USA
    Angew Chem Int Ed Engl 52:9238-41. 2013
    ..The importance of the axial thiolate ligand and the basic nature of compound II ferryl oxygen atom are discussed in terms of these redox potentials. ..
  4. pmc Cytochrome c causes pore formation in cardiolipin-containing membranes
    Chris L Bergstrom
    Department of Chemistry, Princeton University, Princeton, NJ 08544, USA
    Proc Natl Acad Sci U S A 110:6269-74. 2013
    ..Our results suggest that the CL-cyt c interaction may be sufficient to allow cyt c permeation of mitochondrial membranes and that cyt c may contribute to its own escape from mitochondria during apoptosis...
  5. pmc Detection and kinetic characterization of a highly reactive heme-thiolate peroxygenase compound I
    Xiaoshi Wang
    Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States
    J Am Chem Soc 134:12897-900. 2012
    ..All results support the formation of a highly reactive AaeAPO oxoiron(IV) porphyrin radical cation intermediate that is the active oxygen species in these hydroxylation reactions. ..
  6. pmc Catalytic peroxynitrite decomposition improves reperfusion injury after heart transplantation
    Gabor Szabo
    Department of Cardiac Surgery, University of Heidelberg, Heidelberg, Germany
    J Thorac Cardiovasc Surg 143:1443-9. 2012
    ..The present study investigated the effects of the potent peroxynitrite decomposition catalyst FP15 on myocardial and endothelial function after hypothermic ischemia-reperfusion in a heterotopic rat heart transplantation model...
  7. pmc Selective hydroxylation of alkanes by an extracellular fungal peroxygenase
    Sebastian Peter
    Department of Bio and Environmental Sciences, International Graduate School of Zittau, Zittau, Germany
    FEBS J 278:3667-75. 2011
    ..It may accordingly have a role in the bioconversion of natural and anthropogenic alkane-containing structures (including alkyl chains of complex biomaterials) in soils, plant litter, and wood...
  8. pmc Single vesicle observations of the cardiolipin-cytochrome C interaction: induction of membrane morphology changes
    Paul A Beales
    Department of Chemical and Environmental Engineering, Yale University, New Haven, Connecticut 06511, United States
    Langmuir 27:6107-15. 2011
    ..We discuss the possible biological implications of our observations in relation to the structure and function of mitochondria...
  9. pmc Peroxynitrite mediates active site tyrosine nitration in manganese superoxide dismutase. Evidence of a role for the carbonate radical anion
    N Basak Surmeli
    Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA
    J Am Chem Soc 132:17174-85. 2010
    ..The loss of MnSOD activity upon Tyr34 nitration implies that the responsible reagent in vivo is peroxynitrite, acting either directly or through the action of •CO3(-)...
  10. pmc Molecular probes of the mechanism of cytochrome P450. Oxygen traps a substrate radical intermediate
    Harriet L R Cooper
    Department of Chemistry, Princeton University, Princeton, NJ 08544, USA
    Arch Biochem Biophys 507:111-8. 2011
    ..The results could be significant with regard to our understanding of iron-catalyzed C-H hydroxylation, the observation of P450-dependent peroxidation and the development of oxidative stress, especially for CYP2E1...
  11. pmc Mechanisms of peroxynitrite interactions with heme proteins
    Jia Su
    Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA
    Inorg Chem 49:6317-29. 2010
    ..It also induces peroxidase activity with the same temperature profile. This process is suggested to model the apoptotic peroxidation of cardiolipin by cytochrome c...
  12. pmc Direct detection of the oxygen rebound intermediates, ferryl Mb and NO2, in the reaction of metmyoglobin with peroxynitrite
    Jia Su
    Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA
    J Am Chem Soc 131:12979-88. 2009
    ..The results indicate that while oxyMb may reduce the concentration of intracellular NO, it would not eliminate the formation of NO(2) as a decomposition product of peroxynitrite...

Research Grants30

  1. Structural bases of the functions of RNA-protein machines
    THOMAS ARTHUR STEITZ; Fiscal Year: 2013
    ..Also of interest will be the ways in which the structures and properties of RNA molecules can be utilized to carry out various biological functions often analogous to those performed by proteins. ..
  2. DEGENERATIVE AND DEMENTING DISEASES OF AGING
    Stanley B Prusiner; Fiscal Year: 2013
    ..The ultimate goal of all the proposed studies is to define the molecular events that feature in the formation of human prions in order to develop therapeutics that cure the human prion diseases. ..
  3. Reactivity of Manganese and Iron Metalloenzyme Models
    David P Goldberg; Fiscal Year: 2013
    ..Fundamental information regarding the mechanisms of biomimetic HAT, OAT, ET and related reactions will be obtained. ..
  4. Peroxiredoxin 6 as an Anti-oxidant Enzyme
    Aron B Fisher; Fiscal Year: 2013
    ..This information could lead to new approaches to increasing the ability of the lung to tolerate oxidant stress. ..
  5. BIOINORGANIC COPPER COORDINATION CHEMISTRY
    Kenneth D Karlin; Fiscal Year: 2013
    ..Potential long-term applications of this basic research include development of enzyme inhibitors and relevant disease therapeutic strategies. ..
  6. Radical Intermediates of Nitric Oxide Synthase &Myocardial Ischemia Reperfusion
    Ah Lim Tsai; Fiscal Year: 2013
    ..The regulation of these radical intermediates by substrate, cofactors and thiol also are studied, both in vitro and ex vivo, in order to elucidate the underlying disease mechanism of myocardial ischemia and reperfusion injury. ..
  7. HORMONAL REGULATION OF BLOOD PRESSURE
    Michal Laniado Schwartzman; Fiscal Year: 2013
    ..ular tone, in the pathophysiology of hypertension and cardiovascular disease. ..
  8. IPF Fibroblast Phenotype
    Craig A Henke; Fiscal Year: 2013
    ..A major objective of this Program Project is to inform decisions of the IPF Clinical Network by providing information that can be translated into novel therapeutic strategies for IPF. ..
  9. Semi-volatile PCBs: Sources, Exposures, Toxicities
    Larry W Robertson; Fiscal Year: 2013
    ..These data and dietary studies in the last Aim will provide a scientific basis for risk assessment and advice for stakeholders with the ultimate goal to protect highly-exposed individuals and populations. ..
  10. Electron Transfer in Iron and Copper Proteins
    Jay R Winkler; Fiscal Year: 2013
    ..Efforts will focus on oxidative generation of Compounds I and II in cytochromes P450 (Aim 3) and a bacterial enzyme that exhibits NOS-like reactivity (Aim 4). ..
  11. Protein Dynamics in Enzymatic Catalysis
    Robert Callender; Fiscal Year: 2013
    ..The Equipment Core (Core A) supports the specialized comprehensive suite of instrumentation for the Program. The Administrative Core (Core B) administers the Program Project. ..