Electron transfer in the superoxide-generating NADPH oxidase complex reconstituted in vitro.
The superoxide (O2-)-generating NADPH oxidase of phagocytic cells is composed of a membrane-bound flavocytochrome (cytochrome b-559) and three cytosolic components, p47-phox, p67-phox, and the small GTPase rac-1 (or 2). Cytochrome b-559 bears the NADPH binding site and the redox centers (FAD and heme). Electron flow through the redox centers, from NADPH to oxygen, is activated consequent to the assembly of the three 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 cytochrome b-559 and recombinant cytosolic components, activated by the anionic amphiphile, lithium dodecyl sulphate. The NADPH oxidase complex assembled in vitro exhibited: (a) a high steady-state electron flow (165 electrons/heme/s); (b) low stationary levels of FAD and heme reduction (about 10%), and (c) a high rate constant of heme oxidation by oxygen (1720 s-1). Surprisingly, the kinetic properties of NADPH oxidase assembled in a semi-recombinant cell-free system, lacking p47-phox (found to generate significant amounts of O2-), were similar to those 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. The kinetic features of NADPH oxidase assembled in vitro from purified and recombinant components differ considerably from those of solubilized enzyme preparations derived from intact stimulated phagocytes. The fast operation of the cell-free system is best explained by the activation-related facilitation of electron flow at both the FAD-->heme and the heme-->oxygen steps.
Koshkin
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V
Julius Friedrich Cohnheim-Minerva Center for Phagocyte Research, Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Israel.
Lotan
O
O
Pick
E
E
D000818:Animals; D002460:Cell Line; D002474:Cell-Free System; D003573:Cytochrome b Group / Q000302:isolation & purification / Q000378:metabolism*; D004579:Electron Transport; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D019255:NADPH Oxidases / Q000378:metabolism*; D010587:Phagocytosis; D010750:Phosphoproteins / Q000096:biosynthesis / Q000378:metabolism*; D045332:Photosystem II Protein Complex; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D018411:Spodoptera; D013481:Superoxides / Q000378:metabolism*
D003573:Cytochrome b Group; D008081:Liposomes; D010750:Phosphoproteins; D045332:Photosystem II Protein Complex; D011994:Recombinant Proteins; D013481:Superoxides; C019313:cytochrome b559; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1