Covalently Bound Flavin in d‐6‐Hydroxynicotine Oxidase from Arthrobacter oxidans

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Abstract
A substituted riboflavin (HNO‐flavin) was isolated from d‐6‐hydroxynicotine oxidase. It was obtained from flavin peptides by hydrolysis with 6 N HCl 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 8α‐(N‐3‐histidyl)‐riboflavin. Hydrolysis of HNO‐flavin in 6N HCl at 125°C 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.