A spectroscopic and kinetic study of Escherichia coli amine oxidase

Escherichia coli amine oxidase has been overexpressed and characterized spectroscopically and by means of pre-steady state kinetics. The X-band EPR spectrum of E. coli amine oxidase prepared with isotopically pure 63Cu and 65Cu shows superhyperfine contributions of three slightly different nitrogen nuclei. The Q-band spectrum of the enzyme indicates the presence of two different copper signals in an approximate one to one stoichiometry. Furthermore, a signal ascribed to enzyme bound Mn2+ is observed. Both the X-band and Q-band EPR signal of the topasemiquinone as prepared in the presence of the substrate 2-phenylethylamine and KCN show multiple hyperfine lines. The Q-band spectrum of the semiquinone shows that the g-tensor is axial or slightly rhombic. The g-value of gx,y=2.005 is consistent with hydrogen bonding between the 5-CN and/or 2-CO atoms of the topasemiquinone with nearby acid/base groups of the protein. Equilibrium incubation experiments with substrate at different pH values and pre-steady state kinetic analysis indicate the presence of a species absorbing at 400 nm preceding the formation of the aminoquinol and the topasemiquinone intermediate. The amount of topasemiquinone formed in equilibrium is governed by a single acid/base group with pK 9.0, the relation between the amount of 400 nm species and pH being more complex. The 400 nm species is proposed to be the protonated product Schiff-base. The nature of other intermediates of the reductive part of the catalytic cycle is also discussed.