Reaction of 2-thio-FAD-reconstituted p-hydroxybenzoate hydroxylase with hydrogen peroxide. Formation of a covalent flavin-protein linkage.
Hydrogen peroxide reacts with 2-thio-FAD-reconstituted p-hydroxybenzoate hydroxylase to yield a long wavelength intermediate (lambda max = 360, 620 nm) which can be isolated in stable form on removal of excess H2O2. The blue flavin derivative slowly decays in a second peroxide-dependent reaction to yield a new flavin product lacking long wavelength absorbance (lambda max = 408, 472 nm). This final peroxide-modified enzyme binds p-hydroxybenzoate with a 10-fold lower affinity than does the native enzyme; furthermore, substrate binding leads to the inhibition of enzyme reduction by NADPH. Trichloroacetic acid treatment of the final peroxide-modified enzyme results in the quantitative conversion of the bound flavin to free FAD. However, gel filtration of the modified enzyme in guanidine hydrochloride at neutral pH leads to the co-elution of protein and modified flavin. The nondenatured peroxide product reacts rapidly with hydroxylamine to yield 2-NHOH-substituted FAD. These observations indicate that the secondary reaction of peroxide with the blue intermediate from 2-thio-FAD p-hydroxybenzoate hydroxylase results in the formation of an acid-labile covalent flavin-protein linkage within the enzyme active site, involving the flavin C-2 position.
D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000378:metabolism*; D002850:Chromatography, Gel; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives* / Q000378:metabolism; D005420:Flavoproteins / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism*; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D013053:Spectrophotometry
D005420:Flavoproteins; D006898:Hydroxylamines; D005182:Flavin-Adenine Dinucleotide; D019811:Hydroxylamine; C037737:2-thio-FAD; D009249:NADP; D006861:Hydrogen Peroxide; D006899:Mixed Function Oxygenases; D015109:4-Hydroxybenzoate-3-Monooxygenase