A spectroscopic and kinetic study of Escherichia coli amine oxidase

Escherichia coli amine oxidase has been overexpressed and characterized spe ctroscopically and by means of pre-steady state kinetics. The X-band EPR sp ectrum of E. coli amine oxidase prepared with isotopically pure (CU)-C-63 a nd (CU)-C-65 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 pres ence of the substrate 2-phenylethylamine and KCN show multiple hyperfine li nes. The Q-band spectrum of the semiquinone shows that the g-tensor is axia l or slightly rhombic. The g-value of g(x,y) = 2.005 is consistent with hyd rogen bonding between the 5-C=N and/or 2-C=O atoms of the topasemiquinone w ith nearby acid/base groups of the protein. Equilibrium incubation experime nts with substrate at different pH values and pre-steady state kinetic anal ysis indicate the presence of a species absorbing at 400 nm preceding the f ormation of the aminoquinol and the topasemiquinone intermediate. The amoun t of topasemiquinone formed in equilibrium is governed by a single acid/bas e 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 par t of the catalytic cycle is also discussed. (C) 2000 Elsevier Science B.V. All rights reserved.