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 inhibition of this enzyme supports cytostatic antitumor effects of inducible NO synthase. The small R2 subunit of class I ribonucleotide reductases contains a stable free radical tyrosine residue required for activity. This radical 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 allowed 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 protein R2 dimer were obtained at 400 microM 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 nitrated with a high yield, probably owing to its restricted accessibility. Dose-response curves for Tyr122 nitration and loss of the free radical were superimposed. However, protein R2 inactivation was higher than nitration of Tyr122, suggesting that nitration of the nonconserved Tyr62 and Tyr289 might be also of importance for peroxynitrite-mediated inhibition of E. coli protein R2.
Guittet
O
O
Unité 8619, Centre National de la Recherche Scientifique, Université Paris-Sud, Orsay, France.
Decottignies
P
P
Serani
L
L
Henry
Y
Y
Le Maréchal
P
P
Laprévote
O
O
Lepoivre
M
M
D000595:Amino Acid Sequence; D002384:Catalysis / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid; D017124:Conserved Sequence; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D005609:Free Radicals / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008958:Models, Molecular; D008970:Molecular Weight; D009566:Nitrates / Q000378:metabolism* / Q000494:pharmacology; D019159:Nonheme Iron Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D020836:Protein Structure, Quaternary / Q000187:drug effects; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D013053:Spectrophotometry; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D005609:Free Radicals; D009566:Nitrates; D019159:Nonheme Iron Proteins; C039642:peroxynitric acid; C002744:3-nitrotyrosine; D014443:Tyrosine; D012264:Ribonucleotide Reductases