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Cobamides and Ribonucleotide Reduction

XII. THE ELECTRON PARAMAGNETIC RESONANCE SPECTRUM OF "ACTIVE COENZYME B₁₂"

From the ¹ From the Institute for Enzyme Research and the Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706
² From the Department of Biochemistry, College of Medicine, University of Iowa, Iowa City, Iowa 52242

Reaction mixtures containing coenzyme B₁₂ (5'-deoxyadenosylcobalamin), an equivalent concentration of ribonucleotide reductase from Lactobacillus leichmannii, dGTP, and dihydrolipoate were incubated for 6 to 1000 ms at 37° in a rapid reaction apparatus and then freeze-quenched. When the frozen samples were examined by EPR spectroscopy, an EPR signal previously undescribed for coenzyme B₁₂ systems was observed, but the signal was not observed with samples frozen more slowly.

The kinetics of the appearance of this signal closely parallel those of the spectrophotometric changes observed previously in this system by stopped flow techniques. When GTP is substituted for dGTP, the paramagnetic species first accumulates and then disappears with kinetics again following those seen by stopped flow spectroscopy. The maximum spin concentration attained in the presence of dGTP or GTP is somewhat higher than that of the intermediate concentration calculated from the spectrophotometric changes, corresponding to about 60% of the original enzyme or coenzyme concentration, whichever is limiting. Changes in the reaction components affect the concentration of the species observed by EPR and by spectrophotometry in the same way. It is concluded that the EPR spectrum and the changes in absorption spectrum are due to the formation of the same cobamide intermediate of ribonucleotide reduction.

The EPR spectrum has not yet been analyzed in sufficient detail to permit proposal of the structure for the intermediate, but it is considered possible that it may be a B_12r-deoxyadenosyl radical-enzyme complex.

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