Kinetic study of the activation of the neutrophil NADPH oxidase by arachidonic acid. Antagonistic effects of arachidonic acid and phenylarsine oxide

The O-2(-) generating NADPH oxidase complex of neutrophils comprises two se ts of components, namely a membrane-bound heterodimeric flavocytochrome b w hich contains the redox centers of the oxidase and water-soluble proteins o f cytosolic origin which act as activating factors of the flavocytochrome. The NADPH oxidase can be activated in a cell-free system consisting of plas ma membranes and cytosol from resting neutrophils in the presence of GTP ga mma S and arachidonic acid. NADPH oxidase activation is inhibited by phenyl arsine oxide (PAO), a sulfhydryl reagent for vicinal or proximal thiol grou ps. The site of action of PAO was localized by photolabeling in the beta-su bunit of flavocytochrome b [Doussiere, J., Poinas, A, Blais, C., and Vignai s, P. V. (1998) fur. J. Biochem. 251, 649-658]. Moreover, the spin state of heme b is controlled by interaction of arachidonic acid with the flavocyto chrome b [Doussiere, J., Gaillard, J., and Vignais, P. V. (1996) Biochemist ry, 35, 13400-13410]. Here we report that the promoting effect of arachidon ic acid on the activation of NADPH oxidase is due to specific binding of ar achidonic acid to flavocytochrome b. Elicitation of NADPH oxidase activity by arachidonic acid is in part associated with an increased affinity of fla vocytochrome b for Or, an effect that was counteracted by the methyl ester of arachidonic acid. On the other hand, the affinity for NADPH was not affe cted by arachidonic acid. We further demonstrate that PAO antagonizes the e ffect of arachidonic acid on oxidase activation by decreasing the affinity of the oxidase for O-2, but not for NADPH. PAO induced a change in the spin state of heme b, as arachidonic acid does, with, however, some differences in the constraints imposed to the heme. It is concluded that the opposite effects of arachidonic acid and PAO are exerted on the beta-subunit of flav ocytochrome b at two different interacting sites.