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Two paramagnetic species accumulate in the ethanolamine deaminase enzyme during turnover on the substrate, 2-aminopropanol: The S=1/2 cobalt ion in cob(II)alamin and an organic radical derived from the substrate (the substrate radical). The two paramagnets interact to form a relatively weakly-coupled biradical system. The Figure below shows the X-band electron spin echo-detected EPR spectrum of the biradical, which was cryotrapped during steady-state turnover of the enzyme. The relatively weak spin-spin interaction leads to a separation of the dominant Co(II) transition intensity (near g_|_=2.2-2.3) and the radical transitions (near g=2.0). This allows us to selectively probe each paramagnetic center by using high resolution techniques of pulsed-EPR spectroscopy. The initial aims of the cob(II)alamin studies were to identify the in situ nitrogenous axial ligand to the cobalt in ethanolamine deaminase (whether it is the "on-board" dimethylbenzimidazole versus a protein histidine imidazole that has been identified in other B12 coenzyme systems), and to assess our ability to resolve interactions of the corrin ring nitrogen nuclei (N21, N22, N23, N24) with Co(II). |
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