title: Anionic lipid-induced conformational changes in human phagocyte flavocytochrome b precede assembly and activation of the NADPH oxidase complex.
abstract: 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 ∼50% 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.
authors.affiliation: Department of Microbiology, 109 Lewis Hall, Montana State University, Bozeman, MT 59717, USA.
authors.forename: Ross M
authors.initials: RM
authors.lastname: Taylor
authors.forename: Marcia H
authors.initials: MH
authors.lastname: Riesselman
authors.forename: Connie I
authors.initials: CI
authors.lastname: Lord
authors.forename: Jeannie M
authors.initials: JM
authors.lastname: Gripentrog
authors.forename: Algirdas J
authors.initials: AJ
authors.lastname: Jesaitis
mesh_terms: D003573:Cytochrome b Group / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D066298:In Vitro Techniques; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D019255:NADPH Oxidases / Q000737:chemistry* / Q000378:metabolism*; D010586:Phagocytes / Q000187:drug effects* / Q000378:metabolism*; D010712:Phosphatidic Acids / Q000737:chemistry / Q000494:pharmacology*; D011487:Protein Conformation / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects; D013481:Superoxides / Q000378:metabolism
chemical_list: D003573:Cytochrome b Group; D008563:Membrane Lipids; D008565:Membrane Proteins; D010712:Phosphatidic Acids; D013481:Superoxides; C036888:cytochrome b558; D019255:NADPH Oxidases; C086986:CYBA protein, human