Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase

Abstract

Diphenyleneiodonium (DPI) and its analogues have been previously shown to react via a radical mechanism whereby an electron is abstracted from a nucleophile to form a radical, which then adds back to the nucleophile to form covalent adducts [Banks (1966) Chem. Rev. 66, 243-266]. We propose that the inhibition of neutrophil NADPH oxidase by DPI occurs via a similar mechanism. A reduced redox centre in the oxidase could serve as electron donor to DPI, and inhibition would occur after direct phenylation of the redox cofactor, or of adjacent amino acid groups by the DPI radical. In the absence of an activatory stimulus, human neutrophil NADPH-oxidase was not inhibited by DPI. The Ki for time-dependent inhibition by DPI of human neutrophil membrane NADPH oxidase was found to be 5.6 microM. Inhibitory potency of DPI was shown to be directly related to rate of enzyme turnover, indicating the need for a reduced redox centre. Adducts were formed between photoreduced flavin (FAD or FMN) and inhibitor (DPI or diphenyliodonium). These were separated by h.p.l.c. and characterized by absorbance spectroscopy, 1H-n.m.r. and fast-atom-bombardment m.s. and found to have properties consistent with substituted 4a,5-dihydroflavins. After incubation of pig neutrophil membranes with DPI, the quantity of recoverable intact flavin was greatly diminished when NADPH was present to initiate oxidase turnover, indicating that the flavin may be the site of DPI activation. These results may provide a common mechanism of action for iodonium compounds as inhibitors of other flavoenzymes.

Reactive oxygen species generation by the ethylene-bis-dithiocarbamate (EBDC) fungicide mancozeb and its contribution to neuronal toxicity in mesencephalic cells

“…Consistent with this, we showed that the inhibition of NADPH oxidase via three distinctly different inhibitors resulted in an attenuation of extracellular peroxide levels in MZ-treated cells. DPI, a non-specific NADPH oxidase inhibitor, prevents electron transport within the NADPH oxidase multisubunit complex (O'Donnell et al, 1993;Li and Trush, 1998;Doussiere et al, 1999). Because of its action, however, DPI can also inhibit other electron transporters, like nitric oxide synthase (Stuehr et al, 1991), xanthine oxidase (Doussiere and Vignais, 1992), and mitochondrial complex I (Li and Trush, 1998).…”

“…When NADPH oxidase subunits assemble on the cell membrane they can catalyze the electron transfer from NADPH to intermediate ligands, like PQ, which then redox cycle and generate ROS Zhang et al, 2004;BonnehBarkay, 2005b). To investigate the potential involvement of cellular oxidases, like NADPH oxidase, in the extracellular ROS generation by MZ, cultures were pretreated with several different NADPH oxidase inhibitors with differing mechanisms of action (O'Donnell et al, 1993;Stolk et al, 1994;Megyeri et al, 1995;Diatchuk et al, 1997;Li et al, 1998;Doussiere et al, 1999). DPI, apocynin and AEBSF all significantly attenuated H 2 O 2 generation in cultures treated with 30 uM MZ (Figs.…”

Cytotoxicity of paraquat in microglial cells: Involvement of PKCδ- and ERK1/2-dependent NADPH oxidase

“…This compound has been shown to block translocation of the cytosolic subunits to the membrane (Dodd-O and Pearse, 2000). DPI is not a selective inhibitor of NADPH oxidase as it can also inhibit electron transport through the cytochrome b (O'Donnell et al, 1993) as well as other redox cycling reactions. However, DPI has been extensively used as a supporting evidence for the presence of flavan catalyzed oxido/reduction reactions including NADPH oxidase (O'Donnell et al, 1994).…”

“…4C). Due to interference of DPI with MTT, its effect on paraquat-induced cytotoxicity was not determined (O'Donnell et al, 1993). We also tested the effects of gp91ds-tat, a specific peptide inhibitor for NADPH oxidase, (Krotz et al, 2007).…”

FAD is a further essential cofactor of the NAD(P)H and O2‐dependent zeaxanthin‐epoxidase

“…Addition of FAD to the thylakoid suspensions restores the enzyme activity. A further indication for the importance of this flavin during the Zea epoxidation is the strong effect of DPI, an inhibitor of flavin-containing enzymes [21,22]. …”