The distance and relative orientation of the C5 ' methyl group of 5 ' -deoxyadenosine and the substrate radical in vitamin B-12 coenzyme-dependent ethanolamine deaminase from Salmonella typhimurium have been characterized by using X-band two-pulse electron spin-echo envelope modulation (ESEEM) spectroscopy in the disordered solid state. The (S)-2-aminopropanol-generated substrate radical catalytic intermediate was prepared by cryotrapping steady-state mixtures of enzyme in which catalytically exchangeable hydrogen sites in the active site had been labeled by previous turnover on H-2(4)-ethanolamine. Simulation of the time- and frequency-domain ESEEM requires two types of coupled H-2. The strongly coupled 2H has an effective dipole distance (r(eff)) of 2.2 Angstrom, and isotropic coupling constant (A(iso)) of -0.35 MHz. The weakly coupled H-2 has r(eff) = 3.8 Angstrom and A(iso) = 0 MHz. The best H-2 ESEEM time- and frequency-domain simulations are achieved with a model in which the hyperfine couplings arise from one strongly coupled hydrogen site and two equivalent weakly coupled hydrogen sites located on the C5 ' methyl group of 5 ' -deoxyadenosine. This model indicates that the unpaired electron on C1 of the substrate radical and C5 ' are separated by 3.2 Angstrom and are thus at closest contact. The close proximity of C1 and C5 ' indicates that C5 ' of the 5 ' -deoxyadenosyl moiety directly mediates radical migration between cobalt in cobalamin and the substrate/product site over a distance of 5-7 Angstrom in the active site of ethanolamine deaminase.