Differential sensitivity of the tyrosyl radical of mouse ribonucleotide reductase to nitric oxide and peroxynitrite.
Ribonucleotide reductase is essential for DNA synthesis in cycling cells. It has been previously shown that the catalytically competent tyrosyl free radical of its small R2 subunit (R2-Y.) is scavenged in tumor cells co-cultured with macrophages expressing a nitric oxide synthase II activity. We now demonstrate a loss of R2-Y. induced either by .NO or peroxynitrite in vitro. The .NO effect is reversible and followed by an increase in ferric iron release from mouse protein R2. A similar increased iron lability in radical-free, diferric metR2 protein suggests reciprocal stabilizing interactions between R2-Y. and the diiron center in the mouse protein. Scavenging of R2-Y. by peroxynitrite is irreversible and paralleled to an irreversible loss of R2 activity. Formation of nitrotyrosine and dihydroxyphenylalanine was also detected in peroxynitrite-modified protein R2. In R2-overexpressing tumor cells co-cultured with activated murine macrophages, scavenging of R2-Y. following NO synthase II induction was fully reversible, even when endogenous production of peroxynitrite was induced by triggering NADPH oxidase activity with a phorbol ester. Our results did not support the involvement of peroxynitrite in R2-Y. scavenging by macrophage .NO synthase II activity. They confirmed the preponderant physiological role of .NO in the process.
Unité 571, Centre National de la Recherche Scientifique, Bâtiment 430, Université Paris-Sud, France.
O
O
Guittet
B
B
Ducastel
J S
JS
Salem
Y
Y
Henry
H
H
Rubin
G
G
Lemaire
M
M
Lepoivre
D000818:Animals; D018920:Coculture Techniques; D004261:DNA Replication; D005609:Free Radicals; D006861:Hydrogen Peroxide / Q000378:metabolism; D008262:Macrophage Activation; D017737:Macrophages, Peritoneal / Q000201:enzymology; D051379:Mice; D019255:NADPH Oxidases / Q000378:metabolism; D009566:Nitrates / Q000494:pharmacology*; D009569:Nitric Oxide / Q000494:pharmacology*; D012264:Ribonucleotide Reductases / Q000187:drug effects / Q000378:metabolism*; D013054:Spectrophotometry, Atomic; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D005609:Free Radicals; D009566:Nitrates; C066447:tyrosyl radical Y(D); C039642:peroxynitric acid; D009569:Nitric Oxide; D014443:Tyrosine; D006861:Hydrogen Peroxide; D012264:Ribonucleotide Reductases; C108427:ribonucleotide reductase R2 subunit; D019255:NADPH Oxidases