PHOTOAFFINITY-LABELING AND PHOTOINACTIVATION OF THE O-2(-)-GENERATINGOXIDASE OF NEUTROPHILS BY AN AZIDO DERIVATIVE OF FAD

A photoactivable derivative of FAD, 4-[N-(4-azido-2-nitrophenyl)amino] butyryl-FAD (NAP(4)-FAD), was synthesized in a tritiated form with tri tium placed in the NAP(4) moiety of the photoprobe. [H-3]NAP(4)-FAD wa s used to photolabel the putative flavin binding site of the O-2(-)-ge nerating NADPH oxidase located in the plasma membrane of bovine neutro phils. Effective photolabeling required partial deflavination of membr anes, which was achieved by mild treatment with ammonium sulfate added to 50% saturation and 0.05% Triton X-100 for 30 min at 2-4 degrees C. Under these conditions, 40-50% of the oxidase activity was lost, but it could be fully recovered by the addition of nanomolar amounts of FA D (K-M = 10-20 nM). Added FAD could be substituted by [H-3]NAP(4)-FAD in photolabeling experiments. In the dark, [H-3]NAP(4)-FAD bound rever sibly with high affinity to deflavinated neutrophil plasma membranes ( K-d = 50 nM), did not transport electrons, and efficiently inhibited t he FAD-dependent restoration of oxidase activity (K-i = 60 nM). Upon p hotoirradiation of neutrophil plasma membranes in the presence of [H-3 ]NAP(4)-FAD, the nitrene derivative formed bound covalently to a 80-12 0 kDa protein that was identified as the beta-subunit of cytochrome b( 558) by immunodetection and enzymatic deglycosylation. The amount of [ H-3]NAP(4)-FAD covalently incorporated into the beta-subunit of cytoch rome b(558) was 80-90% of the amount of photoprobe specifically bound to neutrophil plasma membranes. A linear relationship between the exte nt of specific photolabeling by [H-3]NAP(4)-FAD and the percentage of NADPH oxidase inactivation was observed for percentages of inactivatio n of up to 70-80%, extrapolating to 0.5 mol of covalently bound [H-3]N AP(4)-FAD per mol of heme b(558).