A photoactivable derivative of FAD, 4-[N-(4-azido-2-nitrophenyl)amino]butyryl-FAD (NAP4-FAD), was synthesized in a tritiated form with tritium placed in the NAP4 moiety of the photoprobe. [3H]NAP4-FAD was used to photolabel the putative flavin binding site of the O2(-)-generating NADPH oxidase located in the plasma membrane of bovine neutrophils. Effective photolabeling required partial deflavination of membranes, 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 FAD (KM = 10-20 nM). Added FAD could be substituted by [3H]NAP4-FAD in photolabeling experiments. In the dark, [3H]NNAP4-FAD bound reversibly with high affinity to deflavinated neutrophil plasma membranes (Kd = 50 nM), did not transport electrons, and efficiently inhibited the FAD-dependent restoration of oxidase activity (Ki = 60 nM). Upon photoirradiation of neutrophil plasma membranes in the presence of [3H]NAP4-FAD, the nitrene derivative formed bound covalently to a 80-120 kDa protein that was identified as the beta-subunit of cytochrome b558 by immunodetection and enzymatic deglycosylation. The amount of [3H]NAP4-FAD covalently incorporated into the beta-subunit of cytochrome b558 was 80-90% of the amount of photoprobe specifically bound to neutrophil plasma membranes. A linear relationship between the extent of specific photolabeling by [3H]NAP4-FAD and the percentage of NADPH oxidase inactivation was observed for percentages of inactivation of up to 70-80%, extrapolating to 0.5 mol of covalently bound [3H]NAP4-FAD per mol of heme b558.