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.

Keywords: cytochrome P450; flavoenzymes; heme; nitric oxide synthase; reactive oxygen species.

FIG. 1.

Effects of diphenyleneiodonium (DPI) on enzymatic activities of microsomes from β-naphthoflavone (NF)–treated rats. Upper panels: cytochrome P450 (CYP) 1A2 and cytochrome c reductase activity in microsomes from β-NF treated rats in the absence or presence of DPI. Center panels: time-dependent inhibition of CYP1A2 and cytochrome c reductase activity. Microsomes were preincubated with DPI and NADPH for 0, 5, and 20 min. Data are the mean ± SE (n = 3). Lower panels: CYP-mediated oxidase activity and cytochrome P450 reductase-mediated menadione redox cycling. Assays were run in the absence or presence of DPI. H₂O₂ formation was measured using the Amplex Red assay.

FIG. 2.

Effects of diphenyleneiodonium (DPI) on cytochrome P450 reductase and cytochrome P450 (CYP) activities in native liver microsomes and recombinant enzymes. All enzyme assays were performed as described in the Materials and Methods. Black symbols represent cytochrome P450 reductase-mediated activities; white symbols represent CYP-mediated activities. Data for NADPH oxidation are results of duplicate measurements. Data in all other assays are the mean ± SE (n = 3). Enzyme reactions were run without preincubations with DPI Enzyme activities included FeCN reduction (red.) and cytochrome c reduction (red.).

FIG. 3.

Effects of diphenyleneiodonium (DPI) on the spectral properties of rat liver microsomes and hemin. Spectra were recorded using liver microsomes from β-naphthoflavone–treated rats (upper panels) and hemin (lower panels). Carbon monoxide (CO) difference spectra (right panels) and binding spectra (left panels) were performed as described in Materials and Methods. CO difference spectra included NADPH as the reductant for rat liver microsomes and sodium dithionite for hemin, respectively. The reference cuvette for CO spectra contained all components in the sample cuvette except CO. Binding spectra for microsomes included NADPH as the reductant; no reductant was used for the hemin binding spectra. The reference cuvette for binding spectra contained all components in the sample cuvette except DPI. Insets: Left panels: time-dependent effects of DPI (1 μM) on the absorbance at the trough (▲, 410 and 366 nm for microsomes and hemin, respectively) and peak (▪, 424 and 426 for microsomes and hemin, respectively) of the binding spectra of rat liver microsomes and hemin. Reactions with microsomes and hemin were initiated with the addition of 100 μM NADPH or DPI, respectively. Upper right panel: dithionite reduced CO spectra in the presence and absence of DPI.

FIG. 4.

Effects of DPI on iNOS. Left panel: the effects of diphenyleneiodonium (DPI) on nitric oxide (NO) production and cytochrome c reduction by iNOS were measured as described in the Materials and Methods. Data are the mean ± SE (n = 3). Right panel: the carbon monoxide (CO) difference spectra of iNOS were recorded in the absence and presence of DPI. Assays were run without preincubations with DPI using sodium dithionite as a reductase.