Exploration of the diaphorase activity of neutrophil NADPH oxidase - Critical assessment of the interaction of iodonitrotetrazolium with the oxidase redox components

In the O-2(-) generating flavocytochrome b, the membrane-bound component of the neutrophil NADPH oxidase, electrons are transported from NADPH to O-2 in the following sequence: NADPH --> FAD --> heme b --> O-2. Although p-iodonitrotetrazolium (INT) has frequently been used as a probe of the diaphorase activity of the neutrophil flavocytochrome b, the propensity of its radical to interact reversibly with O-2 led us to question its specificity. This study was undertaken to reexamine the interaction of TNT with the redox components of the neutrophil flavocytochrome b. Two series of inhibitors were used, namely the flavin analog 5-deaza FAD and the heme inhibitors bipyridyl and benzylimidazole. The following results indicate that TNT reacts preferentially with the hemes rather than with the FAD redox center of flavocytochrome b and is not therefore a specific probe of the diaphorase activity of flavocytochrome b. First, in anaerobiosis, reduced heme b in activated membranes was reoxidized by TNT as efficiently as by O-2 even in the presence of concentrations of 5-deaza FAD which fully inhibited the NADPH oxidase activity. Second, the titration curve of dithionite-reduced heme b in neutrophil membranes obtained by oxidation with increasing amounts of TNT was strictly superimposable on that of dithionite-reduced hemin. Third, TNT competitively inhibited the O-2 uptake by the activated NADPH oxidase in a cell-free system. Finally, the heme inhibitor bipyridyl competitively inhibited the reduction of INT in anaerobiosis, and the oxygen uptake in aerobiosis.