Kinetic and Thermodynamic Characterization of Co^II−Substrate Radical Pair Formation in Coenzyme B₁₂-Dependent Ethanolamine Ammonia-Lyase in a Cryosolvent System by Using Time-Resolved, Full-Spectrum Continuous-Wave Electron Paramagnetic Resonance Spectroscopy

The formation of the Co^II−substrate radical pair catalytic intermediate in coenzyme B₁₂ (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 = τ_obs^-1) of the EPR-detected Co^II−substrate radical pair state. The detection deadtime:  τ_obs ratio is reduced by >10², relative to millisecond rapid mixing experiments at ambient temperatures. The EPR spectrum acquisition time is τ_obs, which allows continuous acquisition of spectra during progress of the reaction. The k_obs values and Co^II−substrate radical pair amplitudes are independent of substrate concentration at each temperature. Therefore, the reaction occurs from the enzyme·coenzyme·substrate ternary complex. The constant value of the Co^II−substrate radical pair amplitude at reaction times >5τ_obs, the approximately 10²-fold slower rate of the substrate radical rearrangement reaction relative to k_obs, and the reversible temperature dependence of the amplitude indicate that the Co^II−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 ΔH₁₃ = 10.8 ± 0.8 kcal/mol and ΔS₁₃ = 45 ± 3 cal/mol/K for the transition from the ternary complex to the Co^II−substrate radical pair state. The free energy difference, ΔG₁₃, is zero to within one standard deviation over the temperature range 234−248 K. The extrapolated value of ΔG₁₃ at 298 K is −2.6 ± 1.2 kcal/mol. The estimated EAL protein-associated contribution to the free energy difference is ΔG_EAL = −24 kcal/mol at 240 K, and ΔH_EAL = −13 kcal/mol and ΔS_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 Co^II−substrate radical pair formation.