The objectives of the proposed work are to contribute to an understanding of the molecular basis for the function of the high valent hemes in unstable peroxidase intermediates using resonance Raman spectroscopy. Structural aspects will be addressed using rapid acquisition resonance Raman techniques which involve the rapid mixing of enzyme, oxidant, and substrate. The products are probed by laser excitation before they can decay into more stable forms. The proposed work will focus on chloroperoxidase from Caladariomyces fumago and yeast cytochrome c peroxidase for which crystallographic data are available.
The aim i s to develop a picture of the interaction of the amino acid residues lining the heme pocket with the activated oxidized hemes in the intermediate states by monitoring variations in specific resonance Raman frequencies as a function of pH and other variables. In cytochrome c peroxidase such effects will be delineated for the oxidized ferryl intermediates using site specific mutations and isotopic substitution. Chloroperoxidase possesses structural and functional similarities to the cytochrome P-450 enzymes. The P-450 enzymes catalyze the hydroxylation of apolar xenobiotics but are also believed to be involved in the activation of carcinogens. A long term goal of the proposed work is to contribute to the understanding of the molecular aspects of the cytochrome P-450 mechanism for which chloroperoxidase intermediates are considered as important models. The chlorinating activity of chloroperoxidase parallels the physiological defense mechanisms of mammalian peroxidases, such as myeloperoxidase and eosinophil peroxidase, which are key components of the immune response. These enzymes can also cause damage to host tissue. Though the peroxidation of chloride ion is central to the activity of these enzymes, the molecular nature of the active oxidizing species is not yet understood.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM057042-01
Application #
2467614
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1998-01-01
Project End
2001-12-31
Budget Start
1998-01-01
Budget End
1998-12-31
Support Year
1
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Virginia Commonwealth University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Richmond
State
VA
Country
United States
Zip Code
23298
Gruia, Flaviu; Ionascu, Dan; Kubo, Minoru et al. (2008) Low-frequency dynamics of Caldariomyces fumago chloroperoxidase probed by femtosecond coherence spectroscopy. Biochemistry 47:5156-67
Kuhnel, Karin; Derat, Etienne; Terner, James et al. (2007) Structure and quantum chemical characterization of chloroperoxidase compound 0, a common reaction intermediate of diverse heme enzymes. Proc Natl Acad Sci U S A 104:99-104
Ward, Kevin R; Barbee, R Wayne; Reynolds, Penny S et al. (2007) Oxygenation monitoring of tissue vasculature by resonance Raman spectroscopy. Anal Chem 79:1514-8
Osborne, Robert L; Coggins, Michael K; Terner, James et al. (2007) Caldariomyces fumago chloroperoxidase catalyzes the oxidative dehalogenation of chlorophenols by a mechanism involving two one-electron steps. J Am Chem Soc 129:14838-9
Weiss, Raymond; Gold, Avram; Terner, James (2006) Cytochromes c': biological models for the S = 3/2, 5/2 spin-state admixture? Chem Rev 106:2550-79
Terner, James; Palaniappan, Vaithianathan; Gold, Avram et al. (2006) Resonance Raman spectroscopy of oxoiron(IV) porphyrin pi-cation radical and oxoiron(IV) hemes in peroxidase intermediates. J Inorg Biochem 100:480-501
Kuhnel, Karin; Blankenfeldt, Wulf; Terner, James et al. (2006) Crystal structures of chloroperoxidase with its bound substrates and complexed with formate, acetate, and nitrate. J Biol Chem 281:23990-8
Schunemann, V; Jung, C; Terner, J et al. (2002) Spectroscopic studies of peroxyacetic acid reaction intermediates of cytochrome P450cam and chloroperoxidase. J Inorg Biochem 91:586-96
Sundaramoorthy, M; Terner, J; Poulos, T L (1998) Stereochemistry of the chloroperoxidase active site: crystallographic and molecular-modeling studies. Chem Biol 5:461-73