Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase.
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.
O'Donnell
B V
BV
Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K.
Tew
D G
DG
Jones
O T
OT
England
P J
PJ
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors* / Q000097:blood; D009249:NADP / Q000378:metabolism / Q000494:pharmacology; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D009861:Onium Compounds / Q000378:metabolism / Q000494:pharmacology*; D013053:Spectrophotometry; D013552:Swine
D007457:Iodine Radioisotopes; D009861:Onium Compounds; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; C007517:diphenyleneiodonium; D005486:Flavin Mononucleotide; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases