ELECTRON-TRANSFER IN THE SUPEROXIDE-GENERATING NADPH OXIDASE COMPLEX RECONSTITUTED IN-VITRO

The superoxide (O-2(-))-generating NADPH oxidase of phagocytic cells i s composed of a membrane-bound flavocytochrome (cytochrome b-559) and three cytosolic components, p47-phox, p67-phox, and the small GTPase r ac-1 (or 2), Cytochrome b-559 bears the NADPH binding site and the red ox centers (FAD and heme). Electron flow through the redox centers, fr om NADPH to oxygen, is activated consequent to the assembly of the thr ee cytosolic components with cytochrome b-559, We studied the kinetics of electron flow through the redox centers of NADPH oxidase in a cell -free system, consisting of purified relipidated and reflavinated cyto chrome b-559 and recombinant cytosolic components, activated by the an ionic amphiphile, lithium dodecyl sulphate. The NADPH oxidase complex assembled in vitro exhibited: (a) a high steady-state electron flow (1 65 electrons/heme/s); (b) low stationary levels of FAD and heme reduct ion (about 10%), and (c) a high rate constant of heme oxidation by oxy gen (1720 s(-1)). Surprisingly, the kinetic properties of NADPH oxidas e assembled in a semi-recombinant cell-free system, lacking p47-phox ( found to generate significant amounts of O-2(-)), were similar to thos e of the complete system, as shown by a steady-state electron flow of 83 electrons/heme/s, low stationary levels of FAD and heme reduction ( 10%), and a rate constant of heme oxidation by oxygen of 1455 s(-1). T he kinetic features of NADPH oxidase assembled in vitro from purified and recombinant components differ considerably from those of solubiliz ed enzyme preparations derived from intact stimulated phagocytes. The fast operation of the cell-fret system is best explained by the activa tion-related facilitation of electron flow at both the FAD --> heme an d the heme --> oxygen steps.