Cytochrome c peroxidase (CcP) is a detoxification enzyme, designed to maintain low intracellular hydrogen peroxide concentrations by reducing the peroxide to water. The catalytic mechanism involves reduction of hydrogen peroxide to water by CcP, in the process oxidizing CcP to an intermediate called CcP compound I (CcP I). CcPI contains an oxidized tryptophan radical as well as an Fe(IV) site. CCP I is reduced back to the native state by ferrocytochrome c. Reduction of CcP I by electron transfer from cytochrome c is the focus of this research proposal. The cytochrome c/CcP system provides an exceptional opportunity to explore details of long-range electron transfer within dynamic, electrostatically-stabilized protein-protein complexes. Structural aspects of the system are well characterized with crystal structures available for CcP and for a number of cytochrome c's from various sources. Crystal structures for 1:1 complexes of yeast iso-1 cytochrome c/CcP and horse cytochrome c/CcP are available. In spite of the exceptional structural information available for the system, the kinetic and mechanistic aspects of the electron transfer processes in the cytochrome c/CcP system are enigmatic. In addition to a high-affinity 1:1 complex of cytochrome c and CcP, recent studies provide definitive evidence for a second, low-affinity cytochrome c-binding site on CcP leading to formation of 2:1 complexes. Most of the controversy surrounding the cytochrome c/CcP system concerns the role of cytochrome c bound to the low-affinity binding site. Two fundamentally different mechanisms have been proposed for electron transfer between cytochrome c and CcP. One mechanism postulates that electron transfer only occurs via the high-affinity binding site while the second postulates that cytochrome c can transfer electrons to the heme site in CcP via both binding domains, with the low-affinity domain actually having the higher electron transfer activity. The primary goal of this proposal is to resolve the role of secondary cytochrome c binding using specific covalent compelxes of CcP and cytochrome c to explore the electron transfer activity of cytochrome c bound to the high- and low-affinity sites on CcP. The covalent complexes will be crosslinked through disulfide bonds using modified CcP and cytochrome c molecules in which cysteine residues are engineered into specific locations on the two proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
1R15GM059740-01
Application #
2884648
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1999-07-01
Project End
2001-06-30
Budget Start
1999-07-01
Budget End
2001-06-30
Support Year
1
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Northern Illinois University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
De Kalb
State
IL
Country
United States
Zip Code
60115
Bidwai, Anil; Ayala, Caitlan; Vitello, Lidia B et al. (2015) Apolar distal pocket mutants of yeast cytochrome c peroxidase: Binding of imidazole, 1-methylimidazole and 4-nitroimidazole to the triAla, triVal, and triLeu variants. Biochim Biophys Acta 1854:919-29
Bidwai, Anil K; Ahrendt, Angela J; Sullivan, John S et al. (2015) pH dependence of cyanide and imidazole binding to the heme domains of Sinorhizobium meliloti and Bradyrhizobium japonicum FixL. J Inorg Biochem 153:88-102
Erman, James E; Vitello, Lidia B; Pearl, Naw May et al. (2015) Binding of Yeast Cytochrome c to Forty-Four Charge-Reversal Mutants of Yeast Cytochrome c Peroxidase: Isothermal Titration Calorimetry. Biochemistry 54:4845-54
Erman, James E; Chinchilla, Diana; Studer, Jason et al. (2015) Binding of imidazole, 1-methylimidazole and 4-nitroimidazole to yeast cytochrome c peroxidase (CcP) and the distal histidine mutant, CcP(H52L). Biochim Biophys Acta 1854:869-81
Chinchilla, Diana; Kilheeney, Heather; Vitello, Lidia B et al. (2014) Kinetic and equilibrium studies of acrylonitrile binding to cytochrome c peroxidase and oxidation of acrylonitrile by cytochrome c peroxidase compound I. Biochem Biophys Res Commun 443:200-4
Bidwai, Anil K; Meyen, Cassandra; Kilheeney, Heather et al. (2013) Apolar distal pocket mutants of yeast cytochrome c peroxidase: hydrogen peroxide reactivity and cyanide binding of the TriAla, TriVal, and TriLeu variants. Biochim Biophys Acta 1834:137-48
Erman, James E; Kilheeney, Heather; Bidwai, Anil K et al. (2013) Peroxygenase activity of cytochrome c peroxidase and three apolar distal heme pocket mutants: hydroxylation of 1-methoxynaphthalene. BMC Biochem 14:19
Foshay, Miriam C; Vitello, Lidia B; Erman, James E (2011) Effect of alternative distal residues on the reactivity of cytochrome c peroxidase: properties of CcP mutants H52D, H52E, H52N, and H52Q. Biochim Biophys Acta 1814:525-35
DiCarlo, Cory M; Vitello, Lidia B; Erman, James E (2011) Reduction potential of yeast cytochrome c peroxidase and three distal histidine mutants: dependence on pH. J Inorg Biochem 105:532-7
Foshay, Miriam C; Vitello, Lidia B; Erman, James E (2009) Relocation of the distal histidine in cytochrome c peroxidase: properties of CcP(W51H), CcP(W51H/H52W), and CcP(W51H/H52L). Biochemistry 48:5417-25

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