The heme component of the neutrophil NADPH oxidase complex is a target for aryliodonium compounds.
The redox core of the neutrophil NADPH oxidase complex is a membrane-bound flavocytochrome b in which FAD and heme b are the two prosthetic redox groups. Both FAD and heme b are able to react with diphenylene iodonium (DPI) and iodonium biphenyl (IBP), two inhibitors of NADPH oxidase activity. In this study, we show that the iodonium modification of heme b contributes predominantly to the inhibition of NADPH oxidase. This conclusion is based on the finding that both iodonium compounds decreased the absorbance of the Soret peak of flavocytochrome b in neutrophil membranes incubated with NADPH, and that this decrease was strictly correlated with the loss of oxidase activity. Furthermore, the heme component of purified flavocytochrome b reduced to no more than 95% by a limited amount of sodium dithionite could be oxidized by DPI or IBP. Butylisocyanide which binds to heme iron precludes heme b oxidation. In activated neutrophil membranes, competitive inhibition of O2 uptake by DPI or IBP occurred transiently and was followed by a noncompetitive inhibition. These results, together with those of EPR spectroscopy experiments, lead us to postulate that DPI or IBP first captures an electron from the reduced heme iron of flavocytochrome b to generate a free radical. Then, the binding of this radical to the proximate environment of the heme iron, most probably on the porphyrin ring, results in inhibition of oxidase activity. In the presence of an excess of sodium dithionite, DPI and IBP produced a biphasic decrease of the Soret band of flavocytochrome b, with a break in the dose effect curve occurring at 50% of the absorbance loss. This was consistent with the presence of two hemes in flavocytochrome b that differ by their sensitivity to DPI or IBP.
Doussiere
J
J
Laboratoire de Biochimie et Biophysique des Systèmes Intégrés (UMR 314 CEA-CNRS), Département de Biologie Moléculaire et Structurale, et Département de Recherche Fondamentale sur la Matière Condensée, SCIB-SCPM, CEA-Grenoble, France.
Gaillard
J
J
Vignais
P V
PV
D000818:Animals; D001713:Biphenyl Compounds / Q000494:pharmacology*; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003573:Cytochrome b Group / Q000737:chemistry / Q000378:metabolism; D004227:Dithionite / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009504:Neutrophils / Q000187:drug effects* / Q000201:enzymology; D009861:Onium Compounds / Q000494:pharmacology*; D010084:Oxidation-Reduction; D010101:Oxygen Consumption / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D001713:Biphenyl Compounds; D003573:Cytochrome b Group; D004791:Enzyme Inhibitors; D009861:Onium Compounds; C031291:diphenyliodonium; D005182:Flavin-Adenine Dinucleotide; D004227:Dithionite; D006418:Heme; C007517:diphenyleneiodonium; C036888:cytochrome b558; D019255:NADPH Oxidases