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A substituted riboflavin (HNO-flavin) was isolated from D-6-hydroxynicotine oxidase. It was obtained from flavin peptides by hydrolysis with 6 N HCI at 95 °C or with aminopeptidase M. The riboflavin derivative had the spectral characteristics of 8α-substituted flavins. It showed a pH dependence of fluorescence with a pK of 4.65 and 86% quenching at pH 7. In thin-layer chromatography it was identical with 8aα-(N-3-histidy1)-riboflavin. Hydrolysis of HNO-flavin in 6N HCI at 125 ° liberated 1 mol histidine per mol flavin as shown by amino acid analysis Since FAD is the coenzyme of D-6-hydroxynicotine oxidase, these results are taken as evidence that this enzyme contains 8α-(N-3-histidyl)-flavin-adenine dinucleotide in the active center. [ABSTRACT FROM AUTHOR]

D-6-Hydroxynicotine oxidase from Arthrobacter oxidans grown on citrate and DL-nicotine was obtained as a homogeneous protein as judged by ultracentrifugation and disc electrophoresis. From the sedimentation and diffusion coefficients of 3.54 S and 6.69 F, respectively, and from sedimentation equilibrium measurements a molecular weight of 53000 was calculated. D-6-Hydroxynicotine oxidase consists of one polypeptide chain and contains one mol FAD per mol enzyme. The flavin was shown to be covalently bound to the protein. In the presence of oxygen, D-6-hydroxynicotine oxidase converts D-6-hydroxynicotine [6-hydroxypyridyl-(3)]-(4-N-methylaminopropyl)-ketone. Molecular oxygen could be replaced as electron acceptor by methylene blue and 2,6-dichlorophenolindophenol but not by one-electron acceptors Anaerobic reduction of the enzyme with substrate did not reveal a semiquinoid flavin intermediate. The spectral changes obtained by the photochemical reduction of D-6-hydroxynicotine oxidase in the presence of EDTA, however, indicated an anionic semiquinone form. D-6-Hydroxynicotine oxidase proved to he highly specific for the structural and absolutely specific for the stereochemical configuration of its substrate. Among others, L-6-hydroxynicotine and the reaction product were found to be competitive inhibitors. [ABSTRACT FROM AUTHOR]