title: Selective Targeting of Heme Protein in Cytochrome P450 and Nitric Oxide Synthase by Diphenyleneiodonium.
abstract: Cytochrome P450 (CYP) enzymes mediate mixed-function oxidation reactions important in drug metabolism. The aromatic heterocyclic cation, diphenyleneiodonium (DPI), binds flavin in cytochrome P450 reductase and inhibits CYP-mediated activity. DPI also inhibits CYP by directly interacting with heme. Herein, we report that DPI effectively inhibits a number of CYP-related monooxygenase reactions including NADPH oxidase, a microsomal enzyme activity that generates hydrogen peroxide in the absence of metabolizing substrates. Inhibition of monooxygenase by DPI was time and concentration dependent with IC50's ranging from 0.06 to 1.9 μM. Higher (4.6-23.9 μM), but not lower (0.06-1.9 μM), concentrations of DPI inhibited electron flow via cytochrome P450 reductase, as measured by its ability to reduce cytochrome c and mediate quinone redox cycling. Similar results were observed with inducible nitric oxide synthase (iNOS), an enzyme containing a C-terminal reductase domain homologous to cytochrome P450 reductase that mediates reduction of cytochrome c, and an N-terminal heme-thiolate oxygenase domain mediating nitric oxide production. Significantly greater concentrations of DPI were required to inhibit cytochrome c reduction by iNOS (IC50 = 3.5 µM) than nitric oxide production (IC50 = 0.16 µM). Difference spectra of liver microsomes, recombinant CYPs, and iNOS demonstrated that DPI altered heme-carbon monoxide interactions. In the presence of NADPH, DPI treatment of microsomes and iNOS yielded a type II spectral shift. These data indicate that DPI interacts with both flavin and heme in CYPs and iNOS. Increased sensitivity for inhibition of CYP-mediated metabolism and nitric oxide production by iNOS indicates that DPI targets heme moieties within the enzymes.
authors.affiliation: *Department of Environmental and Occupational Health, Rutgers University School of Public Health, Piscataway, NJ 08854.
authors.forename: John T
authors.initials: JT
authors.lastname: Szilagyi
authors.affiliation: Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ 08854.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Mishin
authors.affiliation: Department of Environmental Health Science, New York Medical College, Valhalla, NY 10595.
authors.forename: Diane E
authors.initials: DE
authors.lastname: Heck
authors.affiliation: *Department of Environmental and Occupational Health, Rutgers University School of Public Health, Piscataway, NJ 08854.
authors.forename: Yi-Hua
authors.initials: YH
authors.lastname: Jan
authors.affiliation: Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ 08854.
authors.forename: Lauren M
authors.initials: LM
authors.lastname: Aleksunes
authors.affiliation: Department of Pharmaceutical Sciences, Northeast Ohio Medical University, Rootstown, OH 44272.
authors.forename: Jason R
authors.initials: JR
authors.lastname: Richardson
authors.affiliation: Department of Chemistry, Lehigh University, Bethlehem, PA 18015.
authors.forename: Ned D
authors.initials: ND
authors.lastname: Heindel
authors.affiliation: Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ 08854.
authors.forename: Debra L
authors.initials: DL
authors.lastname: Laskin
authors.affiliation: *Department of Environmental and Occupational Health, Rutgers University School of Public Health, Piscataway, NJ 08854 jlaskin@eohsi.rutgers.edu.
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Laskin
mesh_terms: D000818:Animals; D065607:Cytochrome P-450 Enzyme Inhibitors / Q000494:pharmacology*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006418:Heme / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009861:Onium Compounds / Q000494:pharmacology*; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D009861:Onium Compounds; D011994:Recombinant Proteins; D009569:Nitric Oxide; D006418:Heme; C007517:diphenyleneiodonium; D003577:Cytochrome P-450 Enzyme System; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse
keywords: cytochrome P450; flavoenzymes; heme; nitric oxide synthase; reactive oxygen species