Anionic lipid-induced conformational changes in human phagocyte flavocytochrome b precede assembly and activation of the NADPH oxidase complex

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• 作者：Ross M. Taylor¹ ; Marcia H. Riesselman ; Connie I. Lord ; Jeannie M. Gripentrog ; Algirdas J. Jesaitis ; ^{umbaj@montana.edu}
• 关键词：Cytochrome b558/559 ; Phosphatidic acid ; Neutrophils ; Nox2 ; NOX ; Oxidants ; Superoxide
• 刊名：Archives of Biochemistry and Biophysics
• 出版年：2012
• 期刊代码：116_00039861
• 类别：ch
• 出版时间：May, 2012
• 卷：521
• 期：1-2
• 页码：24-31
• 文件大小：624 K

摘要

Phagocyte NADPH oxidases generate superoxide at high rates in defense against infectious agents, a process regulated by second messenger anionic lipids using incompletely understood mechanisms. We reconstituted the catalytic core of the human neutrophil NADPH oxidase, flavocytochrome b (Cyt b) in 99%phosphatidylcholine vesicles in order to correlate anionic lipid-dependent conformational changes in membrane-bound Cyt b and oxidase activity. The anionic lipid 10:0 phosphatidic acid (10:0 PA) specifically induced conformational changes in Cyt b as measured by a combination of fluorescence resonance energy transfer methods and size exclusion chromatography. The fluorescence lifetime of a complex between Cyt b and Cascade Blue-derivatized anti-p22^phox antibody (CCB-CS9), increased after exposure to 10:PA by 鈭?0%of the change observed when the complex is dissociated, indicating a structural rearrangement of p22^phox and/or the Cyt b heme prosthetic groups. Half of the quenching relaxation occurred at 10:0 PA concentrations permissive to less than 10%full NADPH oxidase activity, but saturated near the saturation in activity in a matched cell-free oxidase assay. We conclude that anionic lipids modulate the conformation of Cyt b in the membrane and suggest they may serve to modulate the structure of Cyt b as a control mechanism for the NADPH oxidase.