Pulsed EPR studies of the semiquinone state of copper-containing amine oxidases

The electron spin echo envelope modulation (ESEEM) technique of pulsed EPR spectroscopy was used to further characterize the radical intermediates observed for porcine kidney diamine oxidase and Arthrobacter P1 methylamine oxidase when the enzymes were reduced with substrates in the presence of cyanide under anaerobic conditions. The data obtained show that the trapped radical intermediate states found for both enzymes are closely similar. ESEEM studies provide evidence that at least one nitrogen nucleus is magnetically coupled to the paramagnet. Isotopic substitution experiments using [N-14]-and [N-15]methylamine with Arthrobacter P1 methylamine oxidase establish that this coupled nitrogen nucleus is derived from substrate. Stimulated echo ESEEM data collected at X-band for [N-14]methylamine with enzyme in the presence of cyanide gives rise to a broad peak in the Fourier transform centered at 3.3 MHz. When [N-15]methylamine was used, this spectral component was no longer observed. For porcine kidney diamine oxidase treated with [1,5-N-14(2)]diaminopentane, a similar spectrum with broad features centered at 1.2 and 3.0 MHz was found. Computer simulation of N-14 ESEEM data show that such results are expected when large anisotropic hyperfine interactions dominate the electron-nucleus coupling and a perpendicular ''edge'', in at least one of the powder pattern line shapes, occurs at low frequency. These experiments rule out the possibility that tyrosine or cyanide radicals are responsible for the radical ESR signals observed for substrate-reduced amine oxidase-cyanide complexes and support a mechanism entailing the binding of the amine substrate to the covalently linked quinone cofactor found in both enzymes.