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 ⅐ ) is scavenged in tumor cells co-cultured with macrophages expressing a nitric oxide synthase II activity. We now demonstrate a loss of R2-Y ⅐ induced either by ⅐ NO or peroxynitrite in vitro. The ⅐ 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 ⅐ and the diiron center in the mouse protein. Scavenging of R2-Y ⅐ 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 ⅐ 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 ⅐ scavenging by macrophage ⅐ NO synthase II activity. They confirmed the preponderant physiological role of ⅐ NO in the process.Nitric oxide is a cell-permeable small radical molecule synthesized in diverse organisms by NO synthase enzymes (NOS), 1 which are P450 self-sufficient hemoproteins using Larginine and dioxygen as substrates (reviewed in Ref. 1). In mammals, physiological functions have been attributed to NOS activities in cardiovascular, neural, gastrointestinal, genitourinary and immune systems (2, 3). So called "neuronal" NOS (NOS I) and "endothelial" NOS (NOS III), usually constitutively expressed, are transiently activated by elevation of intracellular Ca 2ϩ concentration (4). A cytokine-inducible NOS (NOS II) is transcriptionally regulated, producing NO at basal Ca 2ϩ levels for up to several days (4). Among cytotoxic and pathophysiological functions, NOS II was rapidly recognized to support a nonspecific antiproliferative activity capable of limiting the growth of invading agents, including viruses, bacteria, parasites, and tumor cells, consistent with the wide distribution of the isoform in different cell types (3, 4).Cytostatic antitumor effector mechanisms of macrophages have been shown to rely on induction of NOS II activity, which severely alters energy production, iron metabolism, and DNA synthesis in tumor target cells (5, 6). There is still a debate about the identity of the cytotoxic nitrogen oxide(s) acting under physiological conditions. For instance, both NO and peroxynitrite ONOO Ϫ have been reported to inhibit mitochondrial respiratory chain, and to regulate iron regulatory protein 1 function, the keystone in cell iron homeostasis (5, 7-10). Ribonucleotide reductase (RR) inhibition by a NOS II product, probably ⅐ NO, has been demonstrated in tumor cells co-cultured with macrop...
