The formation of the Coll-substrate radical pair catalytic intermediate in coenzyme B-12 (adenosylcobalamin)-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium has been studied by using time-resolved continuous-wave electron paramagnetic resonance (EPR) spectroscopy in a cryosolvent system. The 41% v/v DMSO/water cryosolvent allows mixing of holoenzyme and substrate, (S)-2-aminopropanol, at 230 K under conditions of kinetic arrest. Temperature step from 230 to 234-248 K initiates the cleavage of the cobalt-carbon bond and the monoexponential rise (rate constant, k(obs) = tau(-1)(obs)) of the EPR-detected Co-II-substrate radical pair state. The detection deadtime: tau(obs) ratio is reduced by > 10(2), relative to millisecond rapid mixing experiments at ambient temperatures. The EPR spectrum acquisition time is <5 tau(obs), the approximately 10(2)-fold slower rate of the substrate radical rearrangement reaction relative to k(obs), and the reversible temperature dependence of the amplitude indicate that the Coll-substrate radical pair and ternary complex are essentially at equilibrium. The reaction is thus treated as a relaxation to equilibrium by using a linear two-step, three-state mechanism. The intermediate state in this mechanism, the Co-II-5'-deoxyadenosyl radical pair, is not detected by EPR at signal-to-noise ratios of 103, which indicates that the free energy of the Co-II-5'-deoxyadenosyl radical pair state is >3.3 kcal/mol, relative to the Co-II-substrate radical pair. Van't Hoff analysis yields Delta H-13 = 10.8 +/- 0.8 kcal/mol and Delta S-13 = 45 +/- 3 cal/mol/K for the transition from the ternary complex to the Coll-substrate radical pair state. The free energy difference, Delta G(13), is zero to within one standard deviation over the temperature range 234-248 K. The extrapolated value of Delta G(13) at 298 K is -2.6 +/- 1.2 kcal/mol. The estimated EAL protein-associated contribution to the free energy difference is Delta G(EAL) = -24 kcal/mol at 240 K, and Delta H-EAL = -13 kcal/mol and Delta S-EAL = 38 cal/mol/K. The results show that the EAL protein makes both strong enthalpic and entropic contributions to overcome the large, unfavorable cobalt-carbon bond dissociation energy, which biases the reaction in the forward direction of Co-C bond cleavage and Coll-substrate radical pair formation.