Structural changes are induced in human neutrophil cytochrome b by NADPH oxidase activators, LDS, SDS, and arachidonate: intermolecular resonance energy transfer between trisulfopyrenyl-wheat germ agglutinin and cytochrome b(558).
Anionic amphiphiles such as sodium- and lithium dodecyl sulfate (SDS, LDS), or arachidonate (AA) initiate NADPH oxidase and proton channel activation in cell-free systems and intact neutrophils. To investigate whether these amphiphiles exert allosteric effects on cytochrome b, trisulfopyrenyl-labeled wheat germ agglutinin (Cascade Blue-wheat germ agglutinin, CCB-WGA) was used as an extrinsic fluorescence donor for resonance energy transfer (RET) to the intrinsic heme acceptors of detergent-solubilized cytochrome b. In solution, cytochrome b complexed with the CCB-WGA causing a rapid, saturable, carbohydrate-dependent quenching of up to approximately 55% of the steady-state fluorescence. Subsequent additions of SDS, LDS, or AA to typical cell-free oxidase assay concentrations completely relaxed the fluorescence quenching. The relaxation effects were specific, and not caused by dissociation of the CCB-WGA-cytochrome b complex or alterations in the spectral properties of the chromophores. In contrast, addition of the oxidase antagonist, arachidonate methyl ester, caused an opposite effect and was able to partially reverse the activator-induced relaxation. We conclude that the activators induce a cytochrome b conformation wherein the proximity or orientation between the hemes and the extrinsic CCB fluorescence donors has undergone a significant change. These events may be linked to NADPH oxidase assembly and activation or proton channel induction.
Foubert
Thomas R
TR
Department of Microbiology, Montana State University, 109 Lewis Hall, Bozeman, MT 59717-3520, USA.
Burritt
James B
JB
Taylor
Ross M
RM
Jesaitis
Algirdas J
AJ
D016718:Arachidonic Acid / Q000494:pharmacology; D003573:Cytochrome b Group / Q000097:blood*; D004735:Energy Transfer; D020536:Enzyme Activators / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D006418:Heme / Q000378:metabolism; D006801:Humans; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D014909:Wheat Germ Agglutinins / Q000378:metabolism*
D003573:Cytochrome b Group; D020536:Enzyme Activators; D014909:Wheat Germ Agglutinins; D016718:Arachidonic Acid; D012967:Sodium Dodecyl Sulfate; D006418:Heme; C036888:cytochrome b558; C028913:dodecyl sulfate; D019255:NADPH Oxidases