Anionic amphiphile and phospholipid-induced conformational changes in human neutrophil flavocytochrome b observed by fluorescence resonance energy transfer

Abstract

The integral membrane protein flavocytochrome b (Cyt b) comprises the catalytic core of the human phagocyte NADPH oxidase complex and serves to initiate a cascade of reactive oxygen species that participate in the elimination of infectious agents. Superoxide production by the NADPH oxidase complex has been shown to be specifically regulated by the enzymatic generation of lipid second messengers following phagocyte activation. In the present study, a Cyt b-specific monoclonal antibody (mAb 44.1) was labeled with Cascade Blue (CCB) and used in resonance energy transfer (RET) studies probing the effects of a panel of lipid species on the structure of Cyt b. The binding of CCB-mAb 44.1 to immunoaffinity-purified Cyt b was both highly specific and resulted in significant quenching of the steady state donor fluorescence. Titration of the CCB-mAb 44.1:Cyt b complex with the anionic amphiphile lithium dodecyl sulfate (LDS) resulted in a saturable relaxation of fluorescence quenching due to conformational changes in Cyt b at concentrations of the amphiphile required for maximum rates of superoxide production by Cyt b in cell-free assays. Similar results were observed for the anionic amphiphile arachidonic acid (AA), although no relaxation of fluorescence quenching was observed for arachidonate methyl ester (AA-ME). Saturable relaxation of fluorescence quenching was also observed with the anionic, 18:1 phospholipids phosphatidic acid (DOPA) and phosphatidylserine (DOPS), while no relaxation was observed upon addition of the neutral 18:1 lipids phosphatidylcholine (DOPC), phosphatidylethanolamine (DOPE) or diacylglycerol (DAG) at similar levels. Further examination of a variety of phosphatidic acid (PA) species demonstrated DOPA to both potently induce conformational changes in Cyt b and to cause more dramatic conformational changes than PA species with shorter, saturated acyl chains. The data presented in this study support the hypothesis that second messenger lipids, such as AA and PA, directly bind to flavocytochrome b and modulate conformational states relevant to the activation of superoxide production.