title: Reconstitution of flavin-depleted neutrophil flavocytochrome b558 with 8-mercapto-FAD and characterization of the flavin-reconstituted enzyme.
abstract: Cytochrome b558 isolated from human neutrophils was inactive and contained no detectable FAD. However, high NADPH oxidase activity was seen upon reconstitution of the cytochrome with either native FAD or 8-mercapto-FAD in the presence of phospholipids (phosphatidylcholine/phosphatidylethanolamine/phosphatidylinositol/ sphingomyelin/cholesterol, 4:2:1:3:3 (w/w)). Their cell-free superoxide-generating activities were 40.5 and 35.5 mol/s/mol of heme, respectively, which corresponded to 70 and 61% of the original activity of the plasma membranes. Both flavins co-eluted with heme and protein on gel exclusion chromatography. The respective specific flavin content was 6.45 and 7.93 nmol/mg of protein and corresponded to a flavin:heme molar ratio of 0.41 and 0.51 consistent with a 2:1 ratio of heme to flavin. Mixing of 8-mercapto-FAD with flavin-depleted cytochrome b558 caused a red-shift of the flavin absorption maximum from 520 nm to around 560 nm, as has been seen when a variety of other apoflavoprotein dehydrogenases bind this analog. The 8-mercapto-FAD reconstituted into the cytochrome reacted readily with either iodoacetamide (k = 38.8 M-1.min-1) or iodoacetic acid (k = 12.1 M-1.min-1) to give a fluorescence spectrum characteristic of a 8-mercaptoflavin derivative, 8-SCH2CONH2 FAD or 8-SCH2COOH FAD. These results indicate that position 8 of FAD bound to the protein is freely accessible to solvent. These studies support the idea that cytochrome b558 is a flavocytochrome.
authors.affiliation: Department of Biochemistry, Aichi Medical University, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Nisimoto
authors.forename: H
authors.initials: H
authors.lastname: Otsuka-Murakami
authors.forename: D J
authors.initials: DJ
authors.lastname: Lambeth
mesh_terms: D001051:Apoenzymes; D001665:Binding Sites; D015153:Blotting, Western; D002474:Cell-Free System; D003573:Cytochrome b Group / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives* / Q000494:pharmacology; D005420:Flavoproteins / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007460:Iodoacetamide / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D015335:Molecular Probes; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013481:Superoxides / Q000378:metabolism; D014867:Water
chemical_list: D001051:Apoenzymes; D003573:Cytochrome b Group; D005420:Flavoproteins; D008565:Membrane Proteins; D015335:Molecular Probes; D014867:Water; D013481:Superoxides; D005182:Flavin-Adenine Dinucleotide; C077475:8-mercapto-FAD; C036888:cytochrome b558; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D007460:Iodoacetamide