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Volume 270, Number 27, Issue of July 07, pp. 16428-16434, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.

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Reconstitution of Flavin-depleted Neutrophil Flavocytochrome b with 8-Mercapto-FAD and Characterization of the Flavin-reconstituted Enzyme

Yukio Nisimoto , Hidetsugu Otsuka-Murakami , David J. Lambeth

Cytochrome b 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 b 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 Mmin) or iodoacetic acid (k = 12.1 Mmin) to give a fluorescence spectrum characteristic of a 8-mercaptoflavin derivative, 8-SCHCONH FAD or 8-SCHCOOH 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 b is a flavocytochrome.

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