Coordination Chemistry of Copper-Containing Amine Oxidases: Nuclear Magnetic Relaxation Dispersion Studies of Copper Binding, Solvent-Water Exchange, Substrate and Inhibitor Binding, and Protein Aggregation

Abstract

The magnetic field dependence of T1-1 of solvent protons (nuclear magnetic relaxation dispersion (NMRD) profiles) was measured for solutions of the resting (oxidized) forms of amine oxidases from bovine plasma, porcine plasma, porcine kidney, and Arthrobacter PI. These amine oxidases may be divided into two classes on the basis of three factors: the paramagnetic contribution to the proton relaxation rate (T1p-1), the temperature dependence of T1p-1, and the effects of the Cu(II) ligand azide on T1p-1. Specifically, the magnitudes of the paramagnetic contributions are consistent with the presence of at least one liganded water molecule in rapid exchange with bulk solvent water for both classes, but an additional exchangeable water ligand is present in the amine oxidases from porcine plasma and Arthrobacter Pl. This water exchanges rapidly at 25 °1C, but relatively slowly at 5 °C, and is probably displaced upon anion binding. Exogenous ligands, such as azide, are known to bind equatorially to tetragonal Cu(II) ions in all amine oxidases; nonetheless, azide binding only slightly decreases Txof the bovine plasma and porcine kidney amine oxidases but significantly reduces T1p-1 of the porcine plasma protein. This suggests that the additional water molecule is equatorial and contributes to the NMRD profiles of the porcine plasma and Arthrobacter Pl amine oxidases. Substrate has no effect on the NMRD profiles of the bovine plasma enzyme. NMRD measurements were also used to monitor copper incorporation into metal-depleted amine oxidases from porcine and bovine plasma. For both oxidases, the relaxivity (T1p-1 per millimole per liter of protein), specific activity, and the intensity of the principal absorption band are approximately !inear functions of the copper content. Close examination of the diamagnetic contributions to the profiles, which principally relate to the rotational relaxation rate of the large protein molecules, suggests that the plasma amine oxidases tend to associate under the conditions of the present experiments: 0.5 mM protein, pH 7.0, and ionic strength (µ) 0.1. Indeed our data show that NMRD measurements are a sensitive method for detecting self-association of these enzymes under common experimental conditions. © 1991, American Chemical Society. All rights reserved.