Identification of the substrate radical intermediate derived from ethanolamine during catalysis by ethanolamine ammonia-lyase

Abstract

Rapid-mix freeze-quench (RMFQ) methods and electron paramagnetic resonance (EPR) spectroscopy have been used to characterize the steady-state radical in the deamination of ethanolamine catalyzed by adenosylcobalamin (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL). EPR spectra of the radical intermediates formed with the substrates, [1-13C]ethanolamine, [2-13C]ethanolamine, and unlabeled ethanolamine were acquired using RMFQ trapping methods from 10 ms to completion of the reaction. Resolved 13C hyperfine splitting in EPR spectra of samples prepared with [1-13C]ethanolamine and the absence of such splitting in spectra of samples prepared with [2-13C]ethanolamine show that the unpaired electron is localized on C1 (the carbinol carbon) of the substrate. The 13C splitting from C1 persists from 10 ms throughout the time course of substrate turnover, and there was no evidence of a detectable amount of a product like radical having unpaired spin on C2. These results correct an earlier assignment for this radical intermediate [Warncke, K., et al. (1999) J. Am. Chem. Soc. 121, 10522-10528]. The EPR signals of the substrate radical intermediate are altered by electron spin coupling to the other paramagnetic species, cob(II)alamin, in the active site. The dipole-dipole and exchange interactions as well as the 1-13C hyperfine splitting tensor were analyzed via spectral simulations. The sign of the isotropic exchange interaction indicates a weak ferromagnetic coupling of the two unpaired electrons. A Co2+-radical distance of 8.7 A was obtained from the magnitude of the dipole-dipole interaction. The orientation of the principal axes of the 13C hyperfine splitting tensor shows that the long axis of the spin-bearing p orbital on C1 of the substrate radical makes an angle of approximately 98 degrees with the unique axis of the d(z2) orbital of Co2+.

Scheme 1

Scheme 2

Figure 1

A: Comparison of the EPR spectra of the steady-state radical intermediates obtained with a ethanolamine by rapid mix freeze quench (RMFQ) at 10 ms, and b (S)-2-aminopropanol obtained by mixing and manually freezing the sample. For ethanolamine, a, the solution of EAL (0.46 mM active sites) and AdoCbl (0.62 mM) in 10 mM Hepes/NaOH, pH 7.5, was mixed with an equal volume of 8 mM ethanolamine in 10 mM Hepes/NaOH adjusted to pH 7.5. For the sample of (S)-2-aminopropanol, b, the sample contained initially 0.2 mM EAL (active sites), 0.4 mM AdoCbl, and 2 mM (S)-2-aminopropanol in 10 mM Hepes/NaOH adjusted to pH 7.5. (S)-2-Aminopropanol was added last and the sample was frozen in ∼ 20 s by dipping the EPR tube into a chilled slush of isopentane. The spectra were recorded at 77 K. Spectrum a is the average of 8 240 s scans and spectrum b is the average of 4 240 s scans. Spectra were recorded at 9.05 GHz (“free electron” g = 2.0023 at 3230 G) under non-saturating microwave power (4 mW) and a field modulation of 8 G. B: Comparison of experimental, a, and simulated, b, EPR spectra of the EPR spectrum of the steady-state radical obtained with ethanolamine as the substrate. The experimental conditions are given in Figure 1A. The parameters used in the simulation are: J = −53 G; D = −43 G, E= −4 G; Euler angles for the D tensor 0°, 25°, 0°; g tensor of Co²⁺ [2.232, 2.205, 1.975]; A tensor of ⁵⁹Co [7, 4, 109] G; line width of Co²⁺ transitions 25 G (isotropic); A_iso ¹⁴N lower axial ligand 15 G; substrate radical g tensor [2.005, 2.003, 2.002]; A tensor of α-proton [−35, −22, −4] G, Euler angles 140°, −110°, −130°; A_iso of β-H_a 12.5 G; A_iso of β-H_b 5.5 G; A_iso ¹⁴N (ethanolamine) 12 G; line width of radical transitions 12 G (isotropic). As noted in the text, the hyperfine splitting parameters representing unresolved hyperfine structure were included in the simulation to account for the inhomogeneously broadened lines. These parameters were derived from separate simulations of spectra of selectively deuterated samples in D₂O (34).

Figure 2

EPR spectra of RMFQ samples with unlabeled ethanolamine and with isotopomers of (¹³C)ethanolamine. The sample compositions are given in the legend of Figure 1 except for substitution of: b [2-¹³C]ethanolamine ; c [1-¹³C]ethanolamine for a ethanolamine. The reactions were freeze-quenched at 10 ms. Each spectrum represents the average of 6 scans. Other acquisition parameters are given in the Legend of Figure 1.

Figure 3

Time course of the EPR signal of the radical intermediate when either A [1-¹³C] ethanolamine or B [2-¹³C]ethanolamine as the substrate. The respective reaction times when the mixtures were freeze quenched are indicated on the figure. Acquisition parameters are as indicated in the Legend of Figure 1.

Figure 4

Analysis of the ¹³C hyperfine splitting interaction in EPR spectra of the substrate radical during the reaction of 1-¹³C-ethanolamine. The experimental spectrum taken at t = 10 ms is reproduced in a. b: The spectrum calculated using a ¹³C hyperfine tensor [10, 10, 120] G and Euler angles 0°, 98°, 20°. c: The spectrum calculated using a ¹³C hyperfine tensor of [7, 13, 110] G and Euler angles 0°, 98°, 20°. d: The spectrum calculated using the same ¹³C tensor as in d but with Euler angles of 0°, 0°, 0°. The other simulation parameters are the same as given in the Legend of Figure 1.