Analysis of the electron paramagnetic resonance spectrum of a radical intermediate in the coenzyme B(12)-dependent ethanolamine ammonia-lyase catalyzed reaction of S-2-aminopropanol.

The structure of the steady-state radical intermediate in the deamination of S-2-aminopropanol catalyzed by ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium has been probed by electron paramagnetic resonance (EPR) spectroscopy using isotopically labeled forms of the substrate and of the adenosylcobalamin cofactor. Electron spin-spin coupling between the radical, centered on the carbon skeleton of the substrate, and the low-spin Co(2+) in cob(II)alamin (B(12r)) produces a dominant splitting of the EPR signals of both the radical and the Co(2+). Analysis of the exchange and dipole-dipole contributions to the spin-spin coupling indicates that the two paramagnetic centers are separated by approximately 11 A. Experiments with (13)C- and with (2)H-labeled forms of S-2-aminopropanol show that the radical is centered on C1 of the carbon skeleton of the substrate in agreement with an earlier report [Babior, B. M., Moss, T. H., Orme-Johnson, W. H., and Beinert, H., (1974) J. Biol. Chem. 249, 4537-4544]. Experiments with perdeutero-S-2-aminopropanol and [2-(15)N]-perdeutero-S-2-aminopropanol reveal a strong hyperfine splitting from the substrate nitrogen, which indicates that the radical is the initial substrate radical created by abstraction of a hydrogen atom from C1 of S-2-aminopropanol. The strong nitrogen hyperfine splitting further indicates that the amino substituent at C2 is approximately eclipsed with respect to the half-occupied p orbital at C1. Experiments with adenosylcobalamin enriched in (15)N in the dimethylbenzimidazole moiety show that the axial base of the cofactor remains attached to the Co(2+) in a functional steady-state reaction intermediate.