Deflavination of flavo-oxidases by nucleophilic reagents

Using spectroscopic techniques we studied the effect of the nucleophilic re agents cyanide, cyanate and thiocyanate on three flavo-oxidases namely alco hol oxidase (AO), glucose oxidase (GOX) and D-amino acid oxidase (DAOX). Al l three ions, added at concentrations in the mM range, caused release of th e flavin adenine dinucleotide (FAD) co-factors from the enzyme molecules. I n the case of AO this was accompanied by significant conformational perturb ations, which was not observed for GOX and DAOX. As suggested from fluoresc ence, absorption and circular dichroism spectral changes at least one pheno lic hydroxyl group became ionized upon FAD release from AO and a new class of Trp residues, fluorescent only in apo-AO protein, was demasked. (C) 2001 Elsevier Science B.V. All rights reserved.