Deuterium Kinetic Isotope Effects Resolve Low-Temperature Substrate Radical Reaction Pathways and Steps in B12-Dependent Ethanolamine Ammonia-Lyase
The first-order reaction kinetics of the cryotrapped 1, 1, 2, 2-2H4-aminoethanol substrate radical intermediate state in the adenosylcobalamin (B12) -dependent ethanolamine ammonia-lyase (EAL) from Salmonella enterica serovar Typhimurium are measured over 203 \xe2\x80\x93 225 K by using time-resolved, full-spectrum electron paramagnetic resonance (EPR) spectroscopy. The studies target fundamental understanding of the mechanism of EAL, the signature enzyme in ethanolamine utilization (Eut) metabolism associated with microbiome homeostasis and disease conditions in the human gut. Incorporation of 2H in the hydrogen transfer that follows the substrate radical rearrangement step in the substrate radical decay reaction sequence leads to an observed 1H/2H isotope effect of approximately 2 that preserves, with high fidelity, the idiosyncratic piecewise pattern of rate constant versus inverse temperature dependence that was previously reported for 1H-substrate, including monoexponential (T\xe2\x89\xa5220 K) and two distinct biexponential (T=203\xe2\x80\x93219 K) regimes. In the global kinetic model, reaction at T\xe2\x89\xa5220 K proceeds from the substrate radical macrostate, S\xe2\x80\xa2, and at T=203\xe2\x80\x93219 K, along parallel pathways from the two sequential microstates, S1\xe2\x80\xa2 and S2\xe2\x80\xa2, that are distinguished by different protein configurations. Decay from S\xe2\x80\xa2, or S1\xe2\x80\xa2 and S2\xe2\x80\xa2, is rate-determined by radical rearrangement (1H) or by contributions from both radical rearrangement and hydrogen transfer (2H). Non-native direct decay to products from S1\xe2\x80\xa2 is a consequence of the free energy barrier to the native S1\xe2\x80\xa2 \xe2\x86\x92 S2\xe2\x80\xa2 protein configurational transition. At physiological temperatures, this is averted by the fast protein configurational dynamics that guide the S1\xe2\x80\xa2 \xe2\x86\x92 S2\xe2\x80\xa2 transition.
INTRODUCTION<!>Enzyme Preparation.<!>EPR Sample Preparation.<!>EPR Spectroscopy and Kinetics Measurements.<!>Empirical fitting of the substrate radical decay: observed rate constants.<!>Temperature-dependence of the observed rate constants for substrate radical decay.<!>Time-resolved, full-spectrum EPR measurements of the cob(II)alamin-substrate radical pair decay.<!>Temperature-dependence of the observed substrate radical decay rate constants.<!>Dual pathway model for substrate radical decay for T\xe2\x89\xa4214 K.<!>2H-substrate radical decay is partially rate-determined by both hydrogen atom transfer and radical rearrangement over 207\xe2\x80\x93214 K.<!>1H-substrate radical decay is rate-determined by only radical rearrangement over 207\xe2\x80\x93214 K.<!>Arrhenius dependences of 2H- and 1H-substrate radical decay over 207\xe2\x80\x93214 K.<!>Kinetics in the plateau region, T = 217 \xe2\x80\x93 219 K.<!>Kinetic bifurcation, T=219 \xe2\x80\x93 220 K.<!>CONCLUSIONS
<p>The adenosylcobalamin (coenzyme B12) –dependent ethanolamine ammonia-lyase [EAL; EC 4.3.1.7; cobalamin (vitamin B12)-dependent enzyme superfamily]1, 2 is the first in a sequence of enzymes that process aminoethanol in the ethanolamine utilization (Eut) metabolic pathway3 that is associated with microbiome homeostasis,4 and Salmonella enterica and Escherichia coli –induced disease conditions, in the human gut.5–7 Toward characterization of the molecular mechanism of EAL, we have used low-temperature (T), time-resolved, full-spectrum electron paramagnetic resonance (EPR) spectroscopy8 to address aminoalkanol conversion to the corresponding aldehyde and ammonia by EAL.9–12 During steady-state turnover, the cob(II)alamin-substrate radical pair state (S•) accumulates and is cryotrapped in high yield.8 Subsequent T–step initiated decay of S• to products is rate-determined by the chemical step of substrate radical rearrangement (involving 1, 2-migration of the amino group2, 13), which resolves this step for first-order kinetic analysis (Figure 1). The decay of S• has been studied over the T range of 190 – 230 K (natural substrate, aminoethanol)9–11 and 220 – 250 K (non-native substrate, 2-aminopropanol).12 The low-T 2-aminopropanol substrate radical decay proceeds along two pathways: (1) a productive pathway proceeding to diamagnetic products, and (2) a destructive pathway leading to an organic radical species uncoupled from cob(II)alamin. The destructive pathway was proposed to originate from direct decay of the first (S1•) of two sequential substrate radical microstates, because S1• has a protein configuration specialized for trapping the nascent substrate radical, rather than direct, forward reaction to products.12 The succeeding state, S2•, is configured for guiding the radical rearrangement (RR) step and the following hydrogen transfer (HT) step, and is the progenitor for the native, productive path to product radical (P•) and diamagnetic product (PH) states.12 Two sequential protein configurational states, S1• and S2• were also proposed to mediate the biexponential decay of the aminoethanol-generated substrate radical.11 Here, we use deuterium (2H) substitution to selectively slow the HT step of the aminoethanol substrate radical decay in EAL from S. enterica serovar Typhimurium (S. typhimurium), in order to further define the decay pathways from S1• and S2• and to address the role of the HT step in the decay reaction, based on the observed 1H/2H isotope effects (IEobs).</p><p>The Arrhenius dependence of the observed rate constants (kobs) for decay of the natural isotopic abundance, 1H-substrate radical, was previously shown to have a piecewise pattern over the T range of 190 to 295 K.11 The T-dependence is characterized by the following regions, with decreasing T: (1) Monoexponential decay extends from 295 to 220 K (mechanism depicted in Figure 1), indicating that the native free energy landscape persists deep into the cryo-regime. (2) An abrupt kinetic bifurcation leads to biexponential decay starting at 219 K, and a flat T-dependence of slow and fast observed first-order rate constants (kobs,s and kobs,f) over 219–217 K. (3) Following a concave-down kink, the biexponential decay continues along two distinct Arrhenius relations, from 214 to 203 K.11 Turnover of EAL on 1, 1, 2, 2-2H4-aminoethanol (denoted as 2H-substrate) leads to incorporation of 2H into the C5'-methyl group of 5'-deoxyadenosine.14–16 Consequently, the transfer of hydrogen (H•) from the C5' donor to the product C2• radical center acceptor in the HT step proceeds with 2H (Figure 1). Previous work showed that decay of the 2H-substrate radical over 190–207 K proceeds with mean IEobs<2 on the slow and fast phases.10 Larger, steady-state isotope effects (IESS) are measured at room T for deuterium (1H/2H, 7.5)1417 and tritium (1H/3H, 107),13 which were assigned to the HT step.18 The discrepancy between the measured 1H/2H IESS of 7.5 and the value of 25, which is a lower limit predicted from the 1H/3H IESS by using the semi-classical Swain-Schaad theory,19 is a conundrum,13 which is also observed for another adenosylcobalamin-dependent eliminase enzyme, diol dehydratase.20</p><p>Here, we determine the rate constants for decay of the 2H-substrate radical over the T-range of 203–225 K, and compare these to the corresponding observed rate constants for decay of the 1H-substrate radical.11 The relatively small (~2), uniform 1H/2H IEobs is consistent with masking of the intrinsic 1H/2H isotope effect (IEint) for the HT step by non-hydrogen isotope sensitive contributions to the activation free energy barriers. Combined analysis of the T-dependence of observed the 2H- and 1H- substrate decay rate constants over 203–214 K leads to a revised model for the decay reaction, in which both S1• and S2• are progenitors for decay pathways to diamagnetic products. The analysis also identifies the contribution of the HT step to the decay reactions for 2H-substrate. Thus, the chemical step of HT joins the protein configurational change (S1• →S2•) and chemical RR (S• → P•) steps that are accessible to study by low-T, time-resolved EPR spectroscopy.</p><!><p>Enzyme was purified from the Escherichia coli overexpression system that incorporated the cloned S. typhimurium EAL coding sequence21 as described22. The specific activity of purified EAL with aminoethanol as substrate was determined by using the coupled assay with alcohol dehydrogenase and NADH23 (20–30 μmol/min/mg; T= 298 K, P=1 atm).</p><!><p>All chemicals were purchased from commercial sources. The procedure for cryotrapping of the cob(II)alamin-substrate radical pair in EAL and low-T kinetic measurements has been described in detail.8 In brief, reactions were performed in aerobic buffer containing 10 mM potassium phosphate (pH 7.5), on ice, and under dim red safe-lighting, to eliminate photochemical degradation of the coenzyme B12 (adenosylcobalamin, AdoCbl) cofactor. AdoCbl was added to 2-fold molar excess over active sites. Substrate 1, 1, 2, 2-2H4-aminoethanol (Cambridge Isotope Laboratories, Inc., Tewksbury, MA) was present at 100 mM. The final concentration of enzyme in EPR samples was 10 mg/ml, which is equivalent to 20 μM,22 and an active site concentration of 120 μM.24, 25 Holoenzyme and substrate solutions were manually mixed and loaded into an EPR tube (4 mm outer diameter; Wilmad-LabGlass, Vineland, New Jersey), and the tube was immersed in isopentane (T≈140 K; elapsed time, 10–15 s).</p><!><p>EPR spectra were collected by using a Bruker E500 ElexSys EPR spectrometer equipped with a Bruker ER4123 SHQE cavity. Instrumentation and methods for measurements of the substrate radical decay kinetics by EPR have been described in detail.8 Briefly, EPR samples were held at a staging temperature of 160–180 K in the ER4131VT cryostat system in the spectrometer, and temperature was step-increased to the decay measurement values. The time from initiation of the temperature step to the start of acquisition of the first spectrum was 30–60 s. Continuous acquisition of EPR spectra proceeded for the duration of the decay (24 s sweep time; 2.56 ms time constant; sampling interval, 5–60 s, depending on T). Temperature at the sample was determined by using an Oxford Instruments ITC503 temperature controller with a calibrated model 19180 4-wire RTD probe, which has ±0.3 K accuracy over the range of decay measurements. The ER4131VT cryostat/controller system provided a temperature stability of ±0.5 K over the length of the EPR sample cavity. The temperature was therefore stable to ±0.5 K during each run.</p><!><p>For each EPR spectrum in the decay time series, the amplitude of the substrate radical signal was obtained from the difference between peak and trough amplitudes of the substrate radical derivative feature around g≈2.0, with baseline-correction. All data processing programs were written in MATLAB (Mathworks, Natick, MA). The observed decays were fitted to monoexponential (Eq. 1, N=1) or biexponential (Eq. 1, N=2) functions by using the following expression,
(1)A(t)A(0)=∑i=1NAiexp[−kit]</p><p>Where A(t)A(0) is the normalized amplitude, Ai is the normalized component amplitude (∑i=1NAi=1 at t=0), and ki is the first-order rate constant. Additional data collection and averaging has led to changes in the mean k values at some temperatures, relative to earlier reports.9, 10 The empirical fitting of the substrate radical decay curves led to observed rate constants, specified as: kobs (monoexponential, T≥220 K) and kobs,s, kobs,f (biexponential, slow and fast components, T<220 K).</p><!><p>The temperature dependences of the microscopic rate constants were fitted by using the expression from Arrhenius reaction rate theory:26
(2)k(T)=Aexp[−EaRT]
where A (units, s−1), Ea (kcal/mol) and R (1.987 cal/mol/K) are the Arrhenius prefactor, the activation energy and the gas constant, respectively.</p><!><p>The CW-EPR spectrum of the cob(II)alamin-substrate radical pair, that accumulates during turnover on substrate 2H4-aminoethanol, shows a prominent, broad derivative-shaped feature centered at 285 mT that corresponds to the g⊥ region of low-spin (S=1/2) Co2+ in cob(II)alamin, and a narrower feature, centered at 330 mT, that corresponds to the substrate radical (Figure S1).272829
Figure 2 shows the time-dependence of the 2H-substrate radical component at 210 K, following T-step. As for the decay of the 1H-substrate radical,9 no other paramagnetic species were detected during the decay to the diamagnetic product state. Figure 3 shows the decay of the amplitude of the 2H-substrate radical at representative T values corresponding to the different kinetic regimes, over the full range of 203 to 225 K.</p><!><p>The 2H-substrate radical decay exhibited monoexponential kinetics for T≥220 K (observed rate constant, kobs) and biexponential kinetics for T<220 K (kobs,s and kobs,f). The values of kobs, kobs,s and kobs,f, and the component amplitudes, Aobs,s and Aobs,f, are presented in Table S1. Figure 4 shows the Arrhenius plot of the observed decay rate constants over 203 – 225 K for decay of the 2H-substrate radical. The piece-wise form of the Arrhenius plot over 203 – 225 K is comparable with that previously reported for the 1H-substrate radical decay,11 including the following domains: (1) T≥220 K, the decay is monoexponential; (2) 217≤T≤219 K, the decay is biexponential, with constant values of kobs,s and kobs,f to within the respective standard deviations, and constant values of Aobs,s and Aobs,f to within the respective standard deviations; (3) T≤214 K, the decay is biexponential, with descending values of kobs,s and kobs,f with decreasing T, and with approximately constant Aobs,s and Aobs,f values.</p><!><p>The combined Arrhenius plots of the observed decay rate constants for the 2H-substrate radical and 1H-substrate radical911 over the T-range of 203 – 225 K are presented in Figure 5. The relations display a common piecewise pattern, but with a vertical offset, owing to an approximately 2-fold decrease in rate constant for the 2H-substrate radical decay. Previously, the biexponential decay of the aminoethanol 1H-substrate radical at T≤214 K was accounted for by a minimal microscopic kinetic model of a series 3-state, 2-step mechanism, involving a "slow" step of interconversion between two substrate radical states, S1• and S2• followed by a "fast" step of first-order reaction from S2• that formed diamagnetic product, PH (Figure 6A).11 In this series model, S1• and S2• represent different protein configurational states that are distinguished by a protein configurational change, and their interconversion (microscopic rate constants, k12, k21) thus proceeds with no making or breaking of covalent bonds or significant electron orbital rehybridization. The canonical chemical steps of RR and HT (radical rearrangement, hydrogen transfer; Figure 1)114 are collapsed under the single step, S2• → PH. This microscopic model is not consistent with the 2H-substrate radical decay results: Fitting of the Arrhenius dependences requires a significant IE on k12 and k21, for the H-isotope-insensitive S1•, S2• interconversion (Supporting Text, Figure S2, Table S2). Thus, the series model (Figure 6A) does not account for the 1H/2H IEobs on both the slow and fast phases of substrate radical decay.</p><p>In the revised microscopic model (Figure 6B), the substrate radical decays through two pathways at T≤214 K, starting from either S1• (Pathway 1; corresponding to the slow decay component, kobs,s) or from S2• (Pathway 2; corresponding to the fast decay component, kobs,f). This model successfully accounts for the observed 1H/2H IEobs on both kobs,s and kobs,f, because both pathways involve chemical, bond-making/bond-breaking steps. Each pathway proceeds through the same formal sequence of substrate to product radical rearrangement [S1• → P1• or S2• → P2•; forward and reverse microscopic rate constants, kSP,i, kPS,i, respectively, (where i=1, 2)] and subsequent hydrogen transfer, HT (P1• → PH1 or P2• → PH2; microscopic rate constants, kHT, i). The inability to detect P• by EPR spectroscopy places a limit on the ratio of populations, P•/S•, of <10−3,30 which is consistent with the difference in energy between P• and S• of 5–9 kcal/mol, calculated by ab initio methods313233 (corresponding to P•/S• <10−5, over the experimental T-range). This condition allows the steady-state assumption for the population of P• during the decay (dP•/dt≈0), which leads to analytical expressions for kobs,s and kobs,f for S• → P• → PH, in terms of the microscopic rate constants, for Pathways 1 and 2:26
Eq. 3kobs, s=kSP, 11+kPS, 1kHT, 1
Eq. 4kobs, f=kSP, 21+kPS, 2kHT, 2</p><p>The effective first-order rate constants, kobs,s and kobs,f in Eqs. 3 and 4 are consistent with the observed single-exponential decay kinetics of S1• (Pathway 1) and S2• (Pathway 2).</p><p>Over 207–214 K, the amplitudes for decay through Pathway 1 and Pathway 2 are relatively constant (2H, Aobs,f=0.6 ±0.2; 1H, Aobs,f=0.4 ±0.1). In contrast, the ratio of kobs,f/kobs,s increases with decreasing T from 214 to 207 K (2H, kobs,f/kobs,s changes from 2.5 to 6.5; 1H, kobs,f/kobs,s changes from 3.3 to 5.7). These features are consistent with the generation of a constant proportion of S1• and S2• by the cryotrapping procedure, and the presence of this proportion, as the initial state for substrate radical decay, at each T-value. The T-independent slow and fast amplitudes in the 207 – 214 K range, and the divergent behavior of the decay rates and amplitudes outside of this range for both the 2H- and 1H-substrate radical (described below), are not consistent with a significant rate of interconversion of S1• and S2•. This leads to the assumption that k12, k21<<kSP. The decays through Channels 1 and 2 are thus considered to proceed independently, at T≤214 K (Figure 6B).</p><!><p>The 1H/2H IEint on HT2 in EAL is estimated as 25,13 based on a consideration of the 1H/3H IEobs of 107, and the semi-classical Swain-Schaad relation.19 This value is consistent with 1H/2H IE values reported for hydrogen transfers in the adenosylcobalamin-dependent enzymes, methylmalonyl-CoA mutase (≥20)34 and glutamate mutase (28).35 The IEobs over 207–214 K for Pathway 1 (IEobs,s=1.9 ±0.4) and Pathway 2 (IEobs,f=2.2 ±0.1) are an order of magnitude lower than 25. Using the assumptions that: (1) the step, S• → P•, is hydrogen isotope-independent, (2) IEint=25 for HT [kHT(1H)=25kHT(2H], and (3) the IEobs,s=1.9, the ratio of Eq. 3 [(kobs,s(1H)/kobs,s(2H)] gives an estimated kPS,1/kHT,1 for the slow Pathway 1 decay of 0.97 (calculation details, Supporting Information). For the fast Pathway 2 decay, the estimated kPS,2/kHT,2 is 1.3. Thus, kPS and kHT are comparable for the 2H-substrate radical decay, which indicates that both HT and RR contribute to rate determination of the S1• and S2• decays for the 2H-substrate over the range, 207–214 K.</p><!><p>The ratio, kPS/kHT, is calculated for the 1H-substrate radical decay, by using the above assumptions, yielding values of (kPS,1/kHT,1)=0.039 and (kPS,2/kHT,2)=0.053 for decay through Pathways 1 and 2, respectively (calculation details, Supporting Information). Thus, for 1H-substrate radical decay, kPS/kHT<<1, indicating that HT is significantly faster than RR, and Eqs. 3 and 4 become kobs,s≈kSP,1 and kobs,f≈kSP,2. The decay of the 1H-substrate radical over the range 207–214 K is thus considered to be rate-determined by the RR step.</p><!><p>Eqs. 3 and 4 were used to fit the T-dependence of kobs,s and kobs,f for the 2H-substrate radical decay and 1H-substrate radical decay over 207–214 K. For the 1H-substrate decay, in accord with the assumptions, kobs,s=kSP,1 and kobs,f=kSP,2, the corresponding Arrhenius parameters from linear fits of kobs,s and kobs,f represent kSP,1 and kSP,2 (Figure 5, Table S3). For the 2H-substrate radical decay, the T-dependence of kobs,s and kobs,f was accounted for by using Eqs. 3 and 4 and the following assumptions: (1) The corresponding kSP(1H, T) was used to represent kSP(2H, T). (2) Arrhenius expressions for kPS and kHT for Pathways 1 and 2 were used in the ratio, kPS/kHT, with two resulting adjustable parameters, (Ea,HT – Ea,PS) and APS/AHT (calculation details, Supporting Information; values, Table S3). Figure 5 shows linear fits of the 2H substrate radical decay data over 207–214 K. The excellent fits support the model, and the tenets that HT makes: (1) no contribution to 1H-substrate radical decay, and (2) a partial rate-limiting contribution to 2H-substrate radical decay.</p><p>Figure 7 depicts the kinetic relationships among the S•, P• and PH states in terms of intermediate and transition states on a free energy diagram. The diagram shows graphically the origin of the relatively small IEobs: The HT barrier for 1H-substrate radical decay lies below the barrier for 2H-substrate. For the 2H-substrate radical decay, the IEint=25 raises the barrier for HT by 1.3 kcal/mol, which makes it comparable to the barrier for RR.</p><!><p>Over 217–219 K, the 2H-substrate radical decay is well-fit by the biexponential function, and the IEobs,s=2.3 ±0.2 and IEobs,f=2.3 ±0.4 are comparable to those for the 207–214 K region. This suggests that the decay mechanisms for the 1H- and 2H-substrate radicals proposed for the 207–214 K range extend to 217–219 K. However, extrapolation of the linear fits of kobs,s and kobs,f for the 2H- and 1H-substrate radical decays to the 217–219 K range (Figure 5), shows that the measured biexponential decay rate constants lie below the extrapolated values, and by comparable factors, at 217 K: For kobs,s, the ratio of extrapolated/measured values is 1.9 ±0.5 (1H) and 2.1 ±0.4 (2H), and for kobs,f, 1.5 ±0.2 (1H) and 1.4 ±0.2 (2H). The lowered kobs values distinguish 217–219 K as a separate regime of the decay kinetics, as recognized previously.11 In addition, there is a shift in the mean normalized amplitudes from 207–214 K to 217–219 K (Table S1) that favors the fast component. The shift is statistically significant for 1H-substrate [mean Aobs,f,207–214 K = 0.37 ±0.07, mean Aobs,f,217–219 K = 0.60 ±0.04], but just within one standard deviation for 2H-subtrate [mean Aobs,f,207–214 K = 0.59 ±0.16, mean Aobs,f,217–219 K = 0.77 ±0.06].</p><p>Different causes of the relatively small, 1.4- to 2.1-fold lowering of the measured versus extrapolated kobs,s and kobs,f in the 217–219 K region (relative to the extrapolations from low T) have been considered, but a model remains elusive. Configurational relaxation of the protein on the time-scale of radical decay, possibly arising from a kinetic bottleneck to configurational relaxation during cryotrapping, was addressed by allowing samples to partially decay at 217–219 K, followed by T-step to 210 K, and measurement of decay kinetics. In these samples, the decay kinetics characteristic of 210 K (Table S1) were reproduced, which indicates that protein configurational relaxation during cryotrapping is detectably complete, and thus, that there is no additional, irreversible relaxation upon return to 217–219 K. Protein relaxation is also not consistent with the biexponential kinetics for 217–219 K, because relaxation during decay would be expected to produce a distribution of rate constants,36 necessitating a power law or stretched exponential form of the decay curve. The absence of an effect on kobs values from variation of dwell time in the 217–219 K region by changing the rate of cooling during cryotrapping, over a range of milliseconds to seconds, is also inconsistent with a significant time-dependent (dynamic) protein relaxation effect.8</p><!><p>Figure 5 identifies 220 K as the lowest T value, for which the decay is monoexponential, for both the 2H- and the 1H-substrate radicals. The transition from biexponential to monoexponential decay kinetics with increasing T is explained, in the context of the parallel decay pathway model in Figure 6B, by an abrupt decrease in the free energy barrier that separates S1• and S2•. This creates a single substrate radical state, S•, which, however, decays from configurations that correspond to S2• at T>219 K (Figure 6B). The proposed decay from S2• implies an absence of decay from S1•, which is supported by the trend toward a dominant S2• population, with increasing T (k12>k21 at T>219 K). The model implies that kSP,2>kSP,1, and leads to the proposal that S2• represents the native state that enables and conducts the RR reaction, consistent with previous proposals,1112 and as considered further in Conclusions.</p><p>The IEobs values for the monoexponential decays at 220 and 223 K, of 1.9 and 1.8, respectively, are comparable with the values observed over 207–219 K, which suggests that the same basic mechanism and rate-limiting steps at low T values are present in the monoexponential regime: The 1H-substrate radical decay rate is throttled by the RR step, and the 2H-substrate radical decay is rate-limited by contributions from both RR and HT.</p><!><p>The hierarchy of states and pathways identified by the combination of 1H/2H isotope effects and T-dependence is depicted in Figure 8. Three distinct regions of low-T kinetic behavior are resolved: 207 – 214, 217 – 219, and 220 – 225 K. The 207 – 214 K region shows linear, Arrhenius dependence of the rate constants on T, and leads to the principal conclusion of this work: The slow and fast observed decay rate constants (kobs,s, kobs,f) correspond to two parallel pathways of substrate radical reaction, originating from two distinct states, S1• (Pathway 1) and S2• (Pathway 2). The slow and fast decay components of the 1H-substrate radical are both rate-determined by the RR reaction. The slow and fast decay components of the 2H-substrate radical are both rate-determined by partial contributions of RR and HT reactions, because hydrogen transfer with 2H raises the free energy barrier for HT to the level of the barrier for RR. The kobs values at 217–219 K diverge from the extrapolated Arrhenius behavior over 207–214 K. Above 219 K, Arrhenius dependence resumes, but with a single decay component. Through the dramatic changes in the T-dependence of kobs among the different regions, IEobs remains approximately uniform at ~2, indicating that the kinetic mechanisms for 1H- (rate limitation by RR) and 2H- (rate limitation by RR and HT) substrate radical decay are maintained.</p><p>The two-pathway model for reaction of the aminoethanol substrate radical (Figures 6B and 8) aligns with the model proposed for 2-aminopropanol substrate radical decay.12 For the 2-aminopropanol substrate radical, low-T reaction proceeds along a destructive pathway from S1•, to form an uncoupled free radical and cob(II)alamin, and along a parallel productive pathway from S2• to diamagnetic products. The progenitor states were proposed to represent EAL protein configurational states, arranged in series in the native reaction, that are specialized for the distinctive functions of substrate radical capture (S1•) and rearrangement-enabling (S2•), respectively.12 For aminoethanol substrate, the free energy barrier to S1•→S2• protein configuration conversion is relatively low from physiological T values down to 220 K and reaction to form diamagnetic products occurs solely from S2•. Below 220 K, a free energy barrier arises to S1•→S2• conversion, the lifetime of S1• is prolonged, and direct reaction of S1• to form diamagnetic products occurs through a non-native, "spillover" pathway. In EAL at physiological temperatures, this potentially deleterious pathway is averted by the fast protein configurational dynamics that guide the S1• → S2• transition.</p>