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 Co2+ in cob(II)alamin (B12r) produces a dominant splitting of the EPR signals of both the radical and the Co2+. Analysis of the exchange and dipole-dipole contributions to the spin-spin coupling indicates that the two paramagnetic centers are separated by ∼11 Å. Experiments with 13C- and with 2H-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 perdeuteroS-2-aminopropanol and [2-15N]-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 15N in the dimethylbenzimidazole moiety show that the axial base of the cofactor remains attached to the Co2+ in a functional steady-state reaction intermediate.
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