Abstract
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.
Highlights
Given by the tyrosine-free radical of the B2 subunit of signal (4,5)
The small differences in the low temperature EPR signals between these four highly conserved tyrosine-free radical structures canbe explained by slightly different angles of the 8-methylene group in relation to theplane of the aromatic ring of tyrosine, reflecting different conformations of the polypeptide chain around thetyrosines
The pronounced difference in microwave saturation blast cells selected for resistance to hydroxyurea haad30-fold between the E. coli B2 tyrosine radical EPR signal and increased M2 activity compared to extractshyfdrroomxyureathe M2 signal could be due to their different interac- sensitive parent cells
Summary
From the.+Department of Biophysics, Arrhenius Laboratory, University of Stockholm, S-106 91 Stockholm, Sweden a n d the §Medical Nobel Institute, Departmentof Biochemistry I, Karolinska Institutet, S-10401 Stockholm, Sweden. The E. coli and T4 ribonucleotide reductase radicals have both been shown tobe tyrosine radicals with spindensities delocalized in their aromatic rings, probably formed through a loss of an electron Slight differences in their EPR spectra a t 77 K have been ascribed to a small difference in the angle of the aromatic ringin relation to the/3-methylene group of tyrosine (6). The concentration of the specific radical was at center between the bacterial and mammalian ribonu- least 20-fold higher in resistantcells than in sensitive cells It cleotide reductase is observed in the ability to was suggested that the resistance to hydroxyureawas due to regenerate thefree radical structure.In contrast to the a selective overproduction of the complete M2 protein or to. B1 binds hydroxyurea-resistant and -sensitive cells using glycerol gradient centrifugations and pure M1 frcoamlf thymus indicated that the increase inM2 activity in resistant cells can at least in part beexplained by an overproductionof M2 protein
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