Characterization of the free radical of mammalian ribonucleotide reductase.
Mouse fibroblast 3T6 cells, selected for resistance to hydroxyurea, were shown to overproduce protein M2, one of the two nonidentical subunits of mammalian ribonucleotide reductase. Packed resistant cells gave an EPR signal at 77 K very much resembling the signal given by the tyrosine-free radical of the B2 subunit of Escherichia coli ribonucleotide reductase. Also, the M2-specific free radical was shown to be located at a tyrosine residue. Of the known tyrosine-free radicals of ribonucleotide reductases from E. coli, bacteriophage T4 infected E. coli and pseudorabies virus infected mouse L cells, the M2-specific EPR signal is most closely similar to the signal of the T4 radical. The small differences in the low temperature EPR signals between these four highly conserved tyrosine-free radical structures can be explained by slightly different angles of the beta-methylene group in relation to the plane of the aromatic ring of tyrosine, reflecting different conformations of the polypeptide chain around the tyrosines. The pronounced difference in microwave saturation between the E. coli B2 tyrosine radical EPR signal and the M2 signal could be due to their different interactions with unspecific paramagnetic ions or with the antiferromagnetically coupled iron pair, shown to be present in the E. coli enzyme and postulated also for the mammalian enzyme. A difference in the iron-radical center between the bacterial and mammalian ribonucleotide reductase is also observed in the ability to regenerate the free radical structure. In contrast to the B2 radical, the M2 tyrosine free radical could be regenerated by merely adding dithiothreitol in the presence of O2 to a cell extract where the radical had previously been destroyed by hydroxyurea treatment.
D000818:Animals; D002478:Cells, Cultured; D004351:Drug Resistance; D004578:Electron Spin Resonance Spectroscopy; D005347:Fibroblasts / Q000201:enzymology; D005609:Free Radicals; D006918:Hydroxyurea / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D008872:Microwaves; D012264:Ribonucleotide Reductases / Q000378:metabolism*
D005609:Free Radicals; D012264:Ribonucleotide Reductases; D006918:Hydroxyurea