STRUCTURE OF THE TOPA-SEMIQUINONE CATALYTIC INTERMEDIATE OF AMINE OXIDASE AS REVEALED BY MAGNETIC-INTERACTIONS WITH EXCHANGEABLE H-2 AND H-1 NUCLEI

Electron spin echo envelope modulation (ESEEM) and continuous wave-electron nuclear double resonance (CW-ENDOR) spectroscopies have been used selectively to characterize hyperfine interactions of aqueous solvent-exchangeable H-1 and H-2 nuclei with unpaired electron spin density in the topa-semiquinone catalytic intermediate of bovine amine oxidase. The radical was generated by anaerobic reduction of enzyme in (H2O)-H-2 or (H2O)-H-1 buffer with substrate benzylamine in the presence of cyanide ion. H-2 ESEEM spectra display a strong anisotropic hyperfine coupling of rhombic symmetry that is assigned to a single, exchangeable H-2 nucleus bonded alpha to the substrate-derived nitrogen atom that is incorporated into the cofactor during catalysis. Solvent exchange of this hydrogen dramatically influences the CW-electron paramagnetic resonance spectrum. ESEEM spectra show no evidence for deuteroxyl deuteron hyperfine coupling, suggesting that the two semiquinone oxygen atoms are deprotonated. Seven pairs of (H2O)-H-2 exchange-sensitive hyperfine couplings are observed in the H-1 CW-ENDOR spectrum. These couplings are assigned to protons involved in hydrogen bonds to the nitrogen and the two oxygen atoms of the radical. The exchange insensitivity of the H-1 CW-ENDOR matrix line and relatively weak H-2 matrix ESEEM demonstrate that the radical is well-sequestered from solvent water. The presence of a single proton covalently bonded to a trigonal nitrogen atom, and the p(p)i-orbital overlap in the C-N bond, shows that the nuclear and electronic structure of the active carbon center in the topa-semiquinone resembles that of the subsequent iminoquinone intermediate.