Overexpression of recombinant mouse and herpes simplex virus ribonucleotide reductase small subunit (protein R2) has been obtained by using the T7 RNA polymerase expression system. Both proteins, which constitute about 30% of the soluble Escherichia coli proteins, have been purified to homogeneity by a rapid and simple procedure. At this stage, few of the molecules contain the iron-tyrosyl free-radical center necessary for activity; however, addition of ferrous iron and oxygen under controlled conditions resulted in a mouse R2 protein containing 0.8 radical and 2 irons per polypeptide chain. In this reaction, one oxygen molecule was needed to generate each tyrosyl radical. Both proteins had full enzymatic activity. EPR spectroscopy showed that iron-center/radical interactions are considerably stronger in both mouse and viral proteins than in E. coli protein R2. CD spectra showed that the bacterial protein contains 70% alpha-helical structure compared to only about 50% in the mouse and viral proteins. Light absorption spectra between 310 and 600 nm indicate close similarity of the mu-oxo-bridged binuclear iron centers in all three R2 proteins. Furthermore, the paramagnetically shifted iron ligand proton NMR resonances show that the antiferromagnetic coupling and ligand arrangement in the iron center are nearly identical in all three species.

Mammalian ribonucleotide reductase consists of two nonidentical subunits, proteins R1 and R2, each inactive alone. The R1 protein binds the ribonucleotide substrates while the R2 protein contains a binuclear iron center and a tyrosyl free radical, essential for activity. The crystal structures of the corresponding Escherichia coli proteins suggest that the distance from the active site in R1 to the tyrosyl radical buried in R2 is about 35 A. Therefore, an electron pathway was suggested between the active site and the tyrosyl radical. Such a pathway could include a conserved tryptophan on the suggested R1 interaction surface of R2 and a conserved aspartic acid hydrogen bonded both to the tryptophan and to a histidine iron ligand. To find experimental support for such an electron pathway, we have replaced the conserved tryptophan in mouse R2 with phenylalanine or tyrosine and the aspartic acid with alanine. All the mutated R2 proteins were shown to bind metal with the same affinity as native R2 and to form the binuclear iron center. In addition, the W103Y and D266A proteins formed a normal tyrosyl free radical while only low amounts of radical were observed in the W103F protein. Neither the kinetic rate constants nor the equilibrium dissociation constant of the R1/R2 complex was affected by the mutations as shown by BIAcore biosensor technique. However, all mutant R2 proteins were completely inactive in the enzymatic assay, supporting the hypothesis that the tryptophan and aspartic acid residues are important links in an amino acid residue specific long-range electron transfer.

We compared the allosteric regulation and effector binding properties of wild type R1 protein and R1 protein with a mutation in the "activity site" (D57N) of mouse ribonucleotide reductase. Wild type R1 had two effector-binding sites per polypeptide chain: one site (activity site) for dATP and ATP, with dATP-inhibiting and ATP-stimulating catalytic activity; and a second site (specificity site) for dATP, ATP, dTTP, and dGTP, directing substrate specificity. Binding of dATP to the specificity site had a 20-fold higher affinity than to the activity site. In all these respects, mouse R1 resembles Escherichia coli R1. Results with D57N were complicated by the instability of the protein, but two major changes were apparent. First, enzyme activity was stimulated by both dATP and ATP, suggesting that D57N no longer distinguished between the two nucleotides. Second, the two binding sites for dATP both had the same low affinity for the nucleotide, similar to that of the activity site of wild type R1. Thus the mutation in the activity site had decreased the affinity for dATP at the specificity site, demonstrating the interaction between the two sites.All ribonucleotide reduction is allosterically controlled to ensure an appropriate supply of each of the four dNTPs required for DNA replication and repair (1-3). Three different classes of ribonucleotide reductases exist in nature. For all of them the substrate specificity of a single protein is regulated by binding of nucleoside triphosphate effectors to specific sites on the protein to provide the required mixture of dNTPs. In recent years, the allosteric regulation of the different classes was studied in detail in various microorganisms (4 -7). X-ray studies of the R1 protein (NrdA) of the class Ia reductase from Escherichia coli (8, 9) and of the large protein (NrdD) of the class III reductase from phage T4 (10) provided detailed structural information concerning effector binding.All three classes are present in bacteria; in some cases one finds all three in the same organism (6). In contrast, only class Ia is found in higher eukaryotes (1-3, 11). To this class belongs also one of the three reductases of E. coli that has become the prototype of class Ia reductases (12). This reductase consists of two tightly bound homodimeric proteins: the larger R1 protein (coded by the nrdA gene) and the smaller R2 protein (coded by the nrdB gene). R1 is the business end of the enzyme, containing both catalytic and allosteric sites. R2 harbors a tyrosyl radical, located at a specific position of the polypeptide chain, and an oxygen-linked di-iron center. The radical is generated through the interaction of the iron center with oxygen. During catalysis the radical function moves from R2 to a cysteine residue in the catalytic site of R1 (2,13,14) and there provides the activation of the ribonucleotide that is required for the reduction of the ribose moiety (3, 15).Each polypeptide of E. coli R1 contains two allosteric sites: one specificity site capable of binding ATP, dATP, dGTP, or dTTP...

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For enzymatic activity, mouse ribonucleotide reductase must form a heterodimeric complex composed of homodimeric R1 and R2 proteins. Both substrate specificity and overall activity are regulated by the allosteric effectors ATP, dATP, dTTP, and dGTP, which bind to two different sites found on R1, the activity site and the substrate specificity site. We have used biosensor technique to directly observe the effects of these nucleotides on R1/R2 interactions. In the absence of effectors, positive cooperativity was observed with a Hill coefficient of 1.8 and a KD of 0.5 microM. In the presence of dTTP or dGTP, there was no cooperativity and subunit interaction was observed at a much lower R1 concentration. The highest R1/R2 affinity was in the presence of dATP or ATP with KDs of 0.05-0.1 microM. In all experiments, the molar stoichiometry between the subunits was close to 1:1. Our data support a model whereby binding of any of the effectors to the substrate specificity site promotes formation of the R1 dimer, which we believe is prerequisite for binding to the R2 dimer. Additional binding of either ATP (a positive effector) or dATP (a negative effector) to the activity site further increases R1/R2 association. We propose that binding of ATP or dATP to the activity site controls enzyme activity, not by changing the aggregation state of the R1/R2 proteins as proposed earlier, but rather by locally influencing the long range electron transport between the catalytic site of R1 and the tyrosyl free radical of R2.