Analysis of the electron paramagnetic resonance spectrum of a radical intermediate in the coenzyme B12-dependent ethanolamine ammonia-lyase catalyzed reaction of S-2-aminopropanol

Vahe Bandarian, George H. Reed

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Abstract

The structure of the steady-state radical intermediate in the deamination of S-2-aminopropanol catalyzed by ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium has been probed by electron paramagnetic resonance (EPR) spectroscopy using isotopically labeled forms of the substrate and of the adenosylcobalamin cofactor. Electron spin-spin coupling between the radical, centered on the carbon skeleton of the substrate, and the low-spin Co2+ in cob(II)alamin (B12r) produces a dominant splitting of the EPR signals of both the radical and the Co2+. Analysis of the exchange and dipole-dipole contributions to the spin-spin coupling indicates that the two paramagnetic centers are separated by ∼11 Å. Experiments with 13C- and with 2H-labeled forms of S-2-aminopropanol show that the radical is centered on C1 of the carbon skeleton of the substrate in agreement with an earlier report [Babior, B. M., Moss, T. H., Orme-Johnson, W. H., and Beinert, H., (1974) J. Biol. Chem. 249, 4537-4544]. Experiments with perdeuteroS-2-aminopropanol and [2-15N]-perdeutero-S-2-aminopropanol reveal a strong hyperfine splitting from the substrate nitrogen, which indicates that the radical is the initial substrate radical created by abstraction of a hydrogen atom from C1 of S-2-aminopropanol. The strong nitrogen hyperfine splitting further indicates that the amino substituent at C2 is approximately eclipsed with respect to the half-occupied p orbital at C1. Experiments with adenosylcobalamin enriched in 15N in the dimethylbenzimidazole moiety show that the axial base of the cofactor remains attached to the Co2+ in a functional steady-state reaction intermediate.

Original languageEnglish (US)
Pages (from-to)8580-8588
Number of pages9
JournalBiochemistry
Volume41
Issue number27
DOIs
StatePublished - Jul 9 2002
Externally publishedYes

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Ethanolamine Ammonia-Lyase
Electron Spin Resonance Spectroscopy
Paramagnetic resonance
Substrates
Skeleton
Nitrogen
Carbon
Reaction intermediates
Deamination
Salmonella
Experiments
Salmonella typhimurium
Hydrogen
Spectrum Analysis
2-aminopropanol
cobamamide
Spectroscopy
Electrons
Atoms

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{d32e1c21494f4e31bdef15da393cda98,
title = "Analysis of the electron paramagnetic resonance spectrum of a radical intermediate in the coenzyme B12-dependent ethanolamine ammonia-lyase catalyzed reaction of S-2-aminopropanol",
abstract = "The structure of the steady-state radical intermediate in the deamination of S-2-aminopropanol catalyzed by ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium has been probed by electron paramagnetic resonance (EPR) spectroscopy using isotopically labeled forms of the substrate and of the adenosylcobalamin cofactor. Electron spin-spin coupling between the radical, centered on the carbon skeleton of the substrate, and the low-spin Co2+ in cob(II)alamin (B12r) produces a dominant splitting of the EPR signals of both the radical and the Co2+. Analysis of the exchange and dipole-dipole contributions to the spin-spin coupling indicates that the two paramagnetic centers are separated by ∼11 {\AA}. Experiments with 13C- and with 2H-labeled forms of S-2-aminopropanol show that the radical is centered on C1 of the carbon skeleton of the substrate in agreement with an earlier report [Babior, B. M., Moss, T. H., Orme-Johnson, W. H., and Beinert, H., (1974) J. Biol. Chem. 249, 4537-4544]. Experiments with perdeuteroS-2-aminopropanol and [2-15N]-perdeutero-S-2-aminopropanol reveal a strong hyperfine splitting from the substrate nitrogen, which indicates that the radical is the initial substrate radical created by abstraction of a hydrogen atom from C1 of S-2-aminopropanol. The strong nitrogen hyperfine splitting further indicates that the amino substituent at C2 is approximately eclipsed with respect to the half-occupied p orbital at C1. Experiments with adenosylcobalamin enriched in 15N in the dimethylbenzimidazole moiety show that the axial base of the cofactor remains attached to the Co2+ in a functional steady-state reaction intermediate.",
author = "Vahe Bandarian and Reed, {George H.}",
year = "2002",
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T1 - Analysis of the electron paramagnetic resonance spectrum of a radical intermediate in the coenzyme B12-dependent ethanolamine ammonia-lyase catalyzed reaction of S-2-aminopropanol

AU - Bandarian, Vahe

AU - Reed, George H.

PY - 2002/7/9

Y1 - 2002/7/9

N2 - The structure of the steady-state radical intermediate in the deamination of S-2-aminopropanol catalyzed by ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium has been probed by electron paramagnetic resonance (EPR) spectroscopy using isotopically labeled forms of the substrate and of the adenosylcobalamin cofactor. Electron spin-spin coupling between the radical, centered on the carbon skeleton of the substrate, and the low-spin Co2+ in cob(II)alamin (B12r) produces a dominant splitting of the EPR signals of both the radical and the Co2+. Analysis of the exchange and dipole-dipole contributions to the spin-spin coupling indicates that the two paramagnetic centers are separated by ∼11 Å. Experiments with 13C- and with 2H-labeled forms of S-2-aminopropanol show that the radical is centered on C1 of the carbon skeleton of the substrate in agreement with an earlier report [Babior, B. M., Moss, T. H., Orme-Johnson, W. H., and Beinert, H., (1974) J. Biol. Chem. 249, 4537-4544]. Experiments with perdeuteroS-2-aminopropanol and [2-15N]-perdeutero-S-2-aminopropanol reveal a strong hyperfine splitting from the substrate nitrogen, which indicates that the radical is the initial substrate radical created by abstraction of a hydrogen atom from C1 of S-2-aminopropanol. The strong nitrogen hyperfine splitting further indicates that the amino substituent at C2 is approximately eclipsed with respect to the half-occupied p orbital at C1. Experiments with adenosylcobalamin enriched in 15N in the dimethylbenzimidazole moiety show that the axial base of the cofactor remains attached to the Co2+ in a functional steady-state reaction intermediate.

AB - The structure of the steady-state radical intermediate in the deamination of S-2-aminopropanol catalyzed by ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium has been probed by electron paramagnetic resonance (EPR) spectroscopy using isotopically labeled forms of the substrate and of the adenosylcobalamin cofactor. Electron spin-spin coupling between the radical, centered on the carbon skeleton of the substrate, and the low-spin Co2+ in cob(II)alamin (B12r) produces a dominant splitting of the EPR signals of both the radical and the Co2+. Analysis of the exchange and dipole-dipole contributions to the spin-spin coupling indicates that the two paramagnetic centers are separated by ∼11 Å. Experiments with 13C- and with 2H-labeled forms of S-2-aminopropanol show that the radical is centered on C1 of the carbon skeleton of the substrate in agreement with an earlier report [Babior, B. M., Moss, T. H., Orme-Johnson, W. H., and Beinert, H., (1974) J. Biol. Chem. 249, 4537-4544]. Experiments with perdeuteroS-2-aminopropanol and [2-15N]-perdeutero-S-2-aminopropanol reveal a strong hyperfine splitting from the substrate nitrogen, which indicates that the radical is the initial substrate radical created by abstraction of a hydrogen atom from C1 of S-2-aminopropanol. The strong nitrogen hyperfine splitting further indicates that the amino substituent at C2 is approximately eclipsed with respect to the half-occupied p orbital at C1. Experiments with adenosylcobalamin enriched in 15N in the dimethylbenzimidazole moiety show that the axial base of the cofactor remains attached to the Co2+ in a functional steady-state reaction intermediate.

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U2 - 10.1021/bi0201217

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