title: The copper-topaquinone-phenylhydrazine-adduct geometry in Escherichia coli amine oxidase derivatized with phenylhydrazines substituted with 19F-NMR relaxation measurements.
abstract: The copper quinoprotein amine oxidase from Escherichia coli was derivatized with phenylhydrazine, substituted with a F3C group at the ortho, meta, or para position. The derivatization of the topaquinone cofactor was verified by ultraviolet/visible spectroscopy. The reduction (with dithionite) of Cu(II) to Cu(I), which was required to obtain reference samples, was verified by EPR spectroscopy. 19F-NMR spectroscopy was carried out on the derivatized enzyme forms, and the spectra showed the line-broadening effect due to the paramagnetic Cu(II). The distance between the Cu and the mean of the three F positions in the F3C groups was calculated by means of the Solomon-Bloembergen equation for the distance-dependent contribution of CU(II) to the transversal-relaxation time of the F resonance. Assuming that the F3C-phenylhydrazines in the enzyme are always aligned towards the Cu in the same way, four configurations can be envisaged that should be taken into account to determine the topology of the two cofactors. Based on these configurations, two spatial positions were found where the calculated distances triangulated, each of these positions having a symmetry-related counterpart above or below the topaquinone-phenylhydrazine plane. If it is assumed that the geometric positions of the phenylhydrazine and topaquinone moieties in the adduct remain the same in the derivatized enzymes, a number of minimum distances between the Cu and certain atoms in the topaquinone moiety of the adduct can be calculated (1.52 +/- 0.06 nm from the C2-O, 1.30 +/- 0.04 nm from the C4-O, and 1.26 +/- 0.04 nm from the C5-N). However, one of the configurations yields very similar distances between the Cu and the C2-O and C4-O. Therefore, no conclusions can be made with regard to which OH group is closest to the Cu. By application of the same approach to the 19F-NMR data obtained for porcine-plasma marine oxidase [Williams, T J. & Falk, M.C.(1986) J. Biol. Chem. 261, 15949- 15954] we observed substantial differences between the topologies of the cofactors in the two enzymes. Possible reasons for this are discussed.
authors.affiliation: Department of Microbiology and Enzymology, Delft University of Technology, Delft, The Netherlands.
authors.forename: V
authors.initials: V
authors.lastname: Steinebach
authors.forename: G A
authors.initials: GA
authors.lastname: De Jong
authors.forename: S S
authors.initials: SS
authors.lastname: Wijmenga
authors.forename: S
authors.initials: S
authors.lastname: De Vries
authors.forename: J A
authors.initials: JA
authors.lastname: Duine
mesh_terms: D006631:Amine Oxidase (Copper-Containing) / Q000737:chemistry*; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D005462:Fluorine Radioisotopes; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D010659:Phenylhydrazines / Q000737:chemistry*
chemical_list: D005462:Fluorine Radioisotopes; D010659:Phenylhydrazines; D004295:Dihydroxyphenylalanine; C067626:6-hydroxydopa quinone; D006631:Amine Oxidase (Copper-Containing)