P67-phox-mediated NADPH oxidase assembly: imaging of cytochrome b558 liposomes by atomic force microscopy.
NADPH oxidase activity depends on the assembly of the cytosolic activating factors, p67-phox, p47-phox, p40-phox, and Rac with cytochrome b(558). The transition from an inactive to an active oxidase complex induces the transfer of electrons from NADPH to oxygen through cytochrome b(558). The assembly of oxidase complex was studied in vitro after reconstitution in a heterologous cell-free assay by using true noncontact mode atomic force microscopy. Cytochrome b(558) was purified from neutrophils and Epstein-Barr virus-immortalized B lymphocytes and incorporated into liposomes. The effect of protein glycosylation on liposome size and oxidase activity was investigated. The liposomes containing the native hemoprotein purified from neutrophils had a diameter of 146 nm, whereas after deglycosylation, the diameter was reduced to 68 nm, although oxidase activity was similar in both cases. Native cytochrome b(558) was used after purification in reconstitution experiments to investigate the topography of NADPH oxidase once it was assembled. For the first time, atomic force microscopy illustrated conformational changes of cytochrome b(558) during the transition from the inactive to the active state of oxidase; height measurements allow the determination of a size of 4 nm for the assembled complex. In the processes that were studied, p67-phox displayed a critical function; it was shown to be involved in both assembly and activation of oxidase complex while p47-phox proceeded as a positive effector and increased the affinity of p67-phox with cytochrome b(558), and p40-phox stabilizes the resting state. The results suggest that although an oligomeric structure of oxidase machinery has not been demonstrated, allosteric regulation mechanisms may be proposed.
Paclet
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GREPI EA 2938 MENRT laboratoire Enzymologie, CHU Grenoble BP 217, 38043 Grenoble Cedex, France.
Coleman
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AW
Vergnaud
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S
Morel
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F
D002474:Cell-Free System / Q000201:enzymology / Q000648:ultrastructure; D002478:Cells, Cultured; D003573:Cytochrome b Group / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D003600:Cytosol / Q000201:enzymology / Q000648:ultrastructure; D004789:Enzyme Activation; D006031:Glycosylation; D006801:Humans; D008081:Liposomes; D018625:Microscopy, Atomic Force; D019255:NADPH Oxidases / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D009504:Neutrophils / Q000201:enzymology* / Q000378:metabolism / Q000648:ultrastructure; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D011487:Protein Conformation
D003573:Cytochrome b Group; D008081:Liposomes; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; C036888:cytochrome b558; D019255:NADPH Oxidases