EPR Studies on Adenosylcobalamin Enzymes

Electron paramagnetic resonance spectroscopy has proved a valuable tool in the study of AdoCbl-dependent enzymes. AdoCbl itself is EPR-silent, but upon homolysis to form Cbl(II), two spins are formed, one on the cobalt (which now has low-spin d configuration) and one on the organic radical. Typically, the two unpaired electrons remain close enough in the enzymeis active site that they interact with one another to give complex, but informative, EPR spectra. 

Among the first enzymes studied by EPR were ethanolamine deaminase and AdoCbl-dependent ribonucleotide reductase (Babior et al., 1974 Orme-Johnson et ah, 1974). The EPR spectrum of ethanolamine deaminase was extensively characterized in the presence of 2-aminopropanol, which is a slow substrate for the enzyme. It exhibits a broad feature at g = 2.34 attributed to Cbl(II), and a sharp doublet at g = 2.01 attributed to an organic radical. Using isotopically-labeled substrates, the organic radical was identified as the C-1 radical of 2-aminopropanol (Babior et al., 1974). The doublet splitting was attributed to dipolar coupling of the Co(II) spin with the substrate radical and the distance between cobalt and the substrate radical was calculated to be about 6. Thus, these experiments yielded the first structural information on the active site of a Bn enzyme. 

---