ASSESSMENT OF THE FLAVOPROTEIN NATURE OF THE REDOX CORE OF NEUTROPHILNADPH OXIDASE

The latent NADPH oxidase activity of purified cytochrome b(558) from r abbit peritoneal neutrophils was expressed in a cell-free system consi sting of either get-filtrated cytosol from resting neutrophils, or a m ixture of the three cytosolic activation factors, namely p47, p67 and the G protein Rac1. The cell-free system was supplemented with arachid onic acid and GTP gamma S. With gel-filtrated cytosol, the oxidase act ivity was relatively high (22 moles O-2(-)/s/mole heme b in the absenc e of added FAD), and enhanced by less than one fourth upon addition of FAD. In contrast. with the purified cytosolic activation factors the rate of O-2(-) production was low (8 moles O-2(-)/s/mole heme b), and enhanced more than two-fold by a saturating concentration of FAD. The specificity of FAD was demonstrated by the lack of effect of FMN. FAD was determined together with heme b and the oxidase activity in eluate s from a Sephacryl column at the last step of the purification of cyto chrome b(558). In the eluted fraction that contained both the maximal inducible oxidase activity and the highest amount of heme b, the molar amount of FAD was 20 times less than that of heme b. It is concluded that cytochrome b(558) is an NADPH-dependent flavocytochrome oxido-red uctase (NADPH oxidase) in which one part of FAD is firmly bound and an other, loosely attached. On the other hand, there may exist a parallel pathway of electron transfer from NADPH via distinct FAD dehydrogenas e(s) to the heme b component of the NADH oxidase. (C) 1996 Academic Pr ess, Inc.