Direct determination of product radical structure reveals the radical rearrangement pathway in a coenzyme B12-dependent enzyme.

A carbinolamine (1-aminoethan-1-ol-2-yl) structure for the product radical in the CoII product radical pair catalytic intermediate state in coenzyme B12 (adenosylcobalamin)-dependent ethanolamine deaminase from Salmonella typhimurium has been determined by using isotope labeling and techniques of electron paramagnetic resonance (EPR) spectroscopy. The presence of nitrogen is detected from the difference in the EPR line shapes of the product radicals that are cryotrapped during steady-state turnover on either 14N- or 15N-labeled aminoethanol substrate. Three-pulse electron spin-echo envelope modulation (ESEEM) spectroscopy of the product radical labeled with 2H reveals two types of beta-2H hyperfine couplings. A structural model is proposed in which the two beta-2H couplings arise from two C1-C2 product radical rotamer states. The sum of the dihedral angles between the C2 p-orbital axis and C1-Hbeta bonds is 120 degrees , which indicates sp3-hybridization at C1. This confirms the C1 carbinolamine structure. The identification of the carbinolamine product radical indicates that the radical rearrangement in ethanolamine deaminase deviates from the solution elimination reaction pathway and proceeds by migration of the amine from C2 of the substrate radical to C1 of the product radical.