Covalently Bound Flavin in <scp>d</scp>‐6‐Hydroxynicotine Oxidase from <i>Arthrobacter oxidans</i>

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.