Peroxynitrite-mediated nitration of the stable free radical tyrosine residue of the ribonucleotide reductase small subunit

Ribonucleotide reductase activity is rate-limiting for DNA synthesis, and i nhibition of this enzyme supports cytostatic antitumor effects of inducible NO synthase. The small R2 subunit of class I ribonucleotide reductases con tains a stable free radical tyrosine residue required for activity. This ra dical is destroyed by peroxynitrite, which also inactivates the protein and induces nitration of tyrosine residues. In this report, nitrated residues in the E. coli R2 protein were identified by UV-visible spectroscopy, mass spectrometry (ESI-MS), and tryptic peptide sequencing. Mass analysis allowe d the detection of protein R2 as a native dimer with two iron clusters per subunit. The measured mass was 87 032 Da, compared to a calculated value of 87 028 Da. Peroxynitrite treatment preserved the non-heme iron center and the dimeric form of the protein. A mean of two nitrotyrosines per E. coli p rotein R2 dimer were obtained at 400 mu M peroxynitrite. Only 3 out of the 16 tyrosines were nitrated, including the free radical Tyr122. Despite its radical state, that should favor nitration, the buried Tyr122 was not nitra ted with a high yield, probably owing to its restricted accessibility. Dose -response curves for Tyr122 nitration and loss of the free radical were sup erimposed. However, protein R2 inactivation was higher than nitration of,,, suggesting that nitration of the nonconserved Tyr62 and Tyr289 might be al so of importance for Tyr122 peroxynitrite-mediated inhibition of E. coli pr otein R2.