PRODUCTION OF RECOMBINANT CYTOCHROME-B558 ALLOWS RECONSTITUTION OF THE PHAGOCYTE NADPH OXIDASE SOLELY FROM RECOMBINANT PROTEINS

Phagocytic white blood cells contain a multicomponent oxidase that gen erates microbicidal products by catalyzing electron transfer from NADP H to molecular oxygen. Activation of this oxidase requires interaction s of a unique membrane flavocytochrome with the cytosolic proteins p47 phox, p67phox, and p21Rac. This flavocytochrome, designated cytochrome b558, is a heteromer comprising a 22-kDa alpha-subunit (p22phox) and a glycosylated almost-equal-to 91-kDa beta-subunit (gp91phox). Cytochr ome b558 was expressed in Sf9 insect cells coinfected with recombinant baculoviruses carrying cDNAs for p22phox and gp91phox. Membranes of t hese cells contained a b-type cytochrome with a dithionite-reduced min us oxidized difference spectrum similar to that of neutrophil cytochro me b558. The recombinant cytochrome b558 beta-subunit was heterogeneou sly N-glycosylated as demonstrated by its susceptibility to cleavage w ith endoglycosidases F and H. In contrast to the neutrophil cytochrome b558, a portion of the N-linked oligosaccharide was of the high manno se type. Recombinant cytochrome b558 supported superoxide production i n a cell-free assay containing recombinant p47phox, p67phox, and p21Ra c. The enzymatic turnover of the partially purified recombinant cytoch rome b558 and neutrophil cytochrome b558 were similar (almost-equal-to 100-160 mol of superoxide generated/s/mol of cytochrome heme, range o f two experiments) and the native and recombinant cytochromes showed s imilar requirements for NADPH and exogenous FAD. These studies represe nt the first reconstitution of the NADPH oxidase solely from recombina nt proteins and define a model system to explore the structure and fun ction of cytochrome b558.