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

Abstract

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 to [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 be 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.