THE COPPER-TOPAQUINONE-PHENYLHYDRAZINE-ADDUCT GEOMETRY IN ESCHERICHIA-COLI AMINE OXIDASE DERIVATIZED WITH PHENYLHYDRAZINES SUBSTITUTED WITHTRIFLUOROMETHYL GROUPS, AS DETERMINED WITH F-19-NMR RELAXATION MEASUREMENTS

The copper quinoprotein amine oxidase from Escherichia coli was deriva tized with phenylhydrazines substituted with a F3C group at the ortho, meta, or para position. The derivatization of the topaquinone cofacto r was verified by ultraviolet/visible spectroscopy. The reduction (wit h dithionite) of Cu(II) to Cu(I), which was required to obtain referen ce samples, was verified by EPR spectroscopy. F-19-NMR spectroscopy wa s carried out on the derivatized enzyme forms, and the spectra showed the line-broadening effect due to the paramagnetic Cu(II). The distanc e between the Cu and the mean of the three F positions in the F3C grou ps was calculated by means of the Solomon-Bloembergen equation for the distance-dependent contribution of Cu(II) to the transversal-relaxati on time of the F resonance. Assuming that the F3C-phenylhydrazines in the enzyme are always aligned towards the Cu in the same way, four con figurations can be envisaged that should be taken into account to dete rmine the topology of the two cofactors. Based on these configurations , two spatial positions were found where the calculated distances tria ngulated, each of these positions having a symmetry-related counterpar t above or below the topaquinone-phenylhydrazine plane. If it is assum ed 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 to paquinone 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 applicati on of the same approach to the F-19-NMR data obtained for por cine-pla sma amine oxidase [Williams, T. J. & Falk, M. C. (1986) J. Biol. Chem. 261, 15949-15954] we observed substantial differences between the top ologies of the cofactors in the two enzymes. Possible reasons for this are discussed.