Herpes simplex virus ribonucleotide reductase: expression in Escherichia coli and purification to homogeneity of a tyrosyl free radical-containing, enzymatically active form of the 38-kilodalton subunit.

Infection of mammalian cells with herpes simplex virus (HSV) induces a virus-encoded ribonucleotide reductase which is different from the cellular enzyme. This essential viral enzyme consists of two nonidentical subunits of 140 and 38 kilodaltons (kDa) which have not previously been purified to homogeneity. The small subunit of ribonucleotide reductases from other species contains a tyrosyl free radical essential for activity. We have cloned the gene for the small subunit of HSV-1 ribonucleotide reductase into a tac expression plasmid vector. After transfection of Escherichia coli, expression of the 38-kDa protein was detected in immunoblots with a specific monoclonal antibody. About 30 micrograms of protein was produced per liter of bacterial culture. The 38-kDa protein was purified to homogeneity in an almost quantitative yield by immunoaffinity chromatography. It contained a tyrosyl free radical which gave a specific electron paramagnetic resonance spectrum identical to that we have observed in HSV-infected mammalian cells and clearly different from that produced by the E. coli and mammalian ribonucleotide reductases. The recombinant 38-kDa subunit had full activity when assayed in the presence of HSV-infected cell extracts deficient in the native 38-kDa subunit.     

The role of herpes simplex virus ribonucleotide reductase small subunit carboxyl terminus in subunit interaction and formation of iron-tyrosyl center structure.

Herpes simplex virus ribonucleotide reductase consists of two nonidentical subunits, proteins R1 and R2, which are required together for activity. Active R2 protein contains a tyrosyl free radical and a binuclear iron center. A truncated form of the R2 subunit, lacking 7 amino acid residues in the carboxyl terminus, was constructed, overexpressed in Escherichia coli and purified to homogeneity. In the presence of ferrous iron and oxygen, the truncated protein readily generated similar amounts of tyrosyl free radical as the intact protein. However, the radical showed differences in EPR characteristics in the truncated protein compared with the normal one, indicating an altered structural arrangement of the radical relative to the iron center. The truncated R2* protein was completely devoid of binding affinity to the R1 protein, demonstrating that the subunit interaction is totally dependent on the 7 outermost carboxyl-terminal amino acids of protein R2.     

Half-site reactivity of the tyrosyl radical of ribonucleotide reductase from Escherichia coli

A C-terminally truncated form of protein B2, the homodimeric small subunit of ribonucleotide reductase from Escherichia coli, was found as the result of an apparently specific proteolysis. Truncated homodimers contain an intact binuclear iron center and a normal tyrosyl radical but have no binding capacity for the other ribonucleotide reductase subunit, protein B1, and are consequently enzymatically inactive. Heterodimers, consisting of one full-length and one truncated polypeptide, formed spontaneously during a chelation-reconstitution cycle and were easily separated from the two homodimeric variants. The heterodimeric form of B2 shows a weak interaction with the B1 subunit resulting in low enzyme activity. Using heterodimers containing deuterated tyrosine on the full-length side and protonated tyrosine on the truncated side, we could demonstrate that the tyrosyl radical was randomly generated in one or the other of the two polypeptide chains of the heterodimeric B2 subunit. The small subunit of ribonucleotide reductase thus conforms to a half-site reactivity.     

Subunit M2 of mammalian ribonucleotide reductase. Characterization of a homogeneous protein isolated from M2-overproducing mouse cells

The M2 subunit of mammalian ribonucleotide reductase was purified to homogeneity from hydroxyurea-resistant, M2-overproducing mouse cells. The purification procedure involved affinity chromatography on an anti-tubulin antibody-Sepharose column and high performance gel permeation chromatography. The pure protein is a dimer of Mr = 88,000, containing stoichiometric amounts of a non-heme iron center and a tyrosyl free radical. The radical is destroyed by hydroxyurea but can readily be regenerated on incubation of the radical-free protein alone with iron-dithiothreitol in the presence of air. The ability to spontaneously regenerate the tyrosyl radical distinguishes protein M2 from the corresponding subunit of Escherichia coli ribonucleotide reductase, protein B2, but apart from that the two proteins are very similar.     

Reduced forms of the iron-containing small subunit of ribonucleotide reductase from Escherichia coli

The B2 subunit of ribonucleotide reductase from Escherichia coli contains a stable tyrosyl free radical and an antiferromagnetically coupled dimeric iron center with high-spin ferric ions. The tyrosyl radical is an oxidized form of tyrosine-122. This study shows that the B2 protein has a fully reduced state, denoted reduced B2, characterized by a normal nonradical tyrosine-122 residue and a dimeric ferrous iron center. Reduced B2 can be formed either from active B2 by a three-electron reduction in the presence of suitable mediators or from apoB2 by addition of two equimolar amounts of ferrous ions in the absence of oxygen. The oxidized tyrosyl radical and the ferric iron center can be generated from reduced B2 by the admission of air. The tyrosyl radical can be selectively reduced by one-electron reduction in the presence of a suitable mediator, yielding metB2, a form that seems identical with the form resulting from treatment of active B2 with hydroxyurea. 1H NMR was used to characterize the paramagnetically shifted resonances associated with the reduced iron center. Prominent resonances were observed around 45 ppm (nonexchangeable with solvent) and 57 ppm (exchangeable with solvent) at 37 degrees C. From the temperature dependence of the chemical shifts of these resonances it was concluded that the ferrous ions in reduced B2 are only weakly, if at all, antiferromagnetically coupled. By comparison with data on the similar iron center of deoxyhemerythrin it is suggested that the 57 ppm resonance should be assigned to protons in histidine ligands of the iron center.     

Reduction of the Fe(III)-tyrosyl radical center of Escherichia coli ribonucleotide reductase by dithiothreitol

The active form of protein B2, the small subunit of ribonucleotide reductase from Escherichia coli, contains a binuclear ferric center and a free radical localized to tyrosine 122 of the polypeptide chain. MetB2 is an inactive form that lacks the tyrosine radical but retains the Fe(III) center. We earlier reported (Fontecave, M., Eliasson, R., and Reichard, P. (1989) J. Biol. Chem. 264, 9164-9170) that enzymes from E. coli interconvert B2 and metB2, possibly as part of a regulatory mechanism. Introduction of the tyrosyl radical into metB2 occurred in two steps: first, the Fe(III) center was reduced to Fe(II), generating "reduced B2"; next oxygen regenerated non-enzymatically both Fe(III) and the tyrosyl radical. Here we demonstrate that dithiothreitol (DTT) between pH 8 and 9.5 also slowly converts metB2 to B2 in the presence of oxygen. Also in this case the reaction occurs stepwise with reduced B2 as an intermediate. DTT reduces Fe(III) of both metB2 and B2. In the latter case this reaction is accompanied by the immediate loss of the tyrosyl radical. Our results indicate that the tyrosyl radical can exist only in the presence of an intact Fe(III) center. In reduced B2 iron is loosely bound to the protein, dissociates on standing and is readily removed by chelating agents. Binding decreases at higher pH. Loss of iron from reduced B2 explains why ferrous iron stimulates and iron chelators inhibit reactivation of metB2. We propose that the reactivation of mammalian ribonucleotide reductase by DTT (Thelander, M., Gr?slund, A., and Thelander, L. (1983) Biochem. Biophys. Res. Commun. 110, 859-865) may proceed via a mechanism similar to the one found here for E. coli protein B2.     

Rnr4p, a novel ribonucleotide reductase small-subunit protein.

Ribonucleotide reductases catalyze the formation of deoxyribonucleotides by the reduction of the corresponding ribonucleotides. Eukaryotic ribonucleotide reductases are alpha2beta2 tetramers; each of the larger, alpha subunits possesses binding sites for substrate and allosteric effectors, and each of the smaller, beta subunits contains a binuclear iron complex. The iron complex interacts with a specific tyrosine residue to form a tyrosyl free radical which is essential for activity. Previous work has identified two genes in the yeast Saccharomyces cerevisiae, RNR1 and RNR3, that encode alpha subunits and one gene, RNR2, that encodes a beta subunit. Here we report the identification of a second gene from this yeast, RNR4, that encodes a protein with significant similarity to the beta-subunit proteins. The phenotype of rnr4 mutants is consistent with that expected for a defect in ribonucleotide reductase; rnr4 mutants are supersensitive to the ribonucleotide reductase inhibitor hydroxyurea and display an S-phase arrest at their restrictive temperature. rnr4 mutant extracts are deficient in ribonucleotide reductase activity, and this deficiency can be remedied by the addition of exogenous Rnr4p. As is the case for the other RNR genes, RNR4 is induced by agents that damage DNA. However, Rnr4p lacks a number of sequence elements thought to be essential for iron binding, and mutation of the critical tyrosine residue does not affect Rnr4p function. These results suggest that Rnr4p is catalytically inactive but, nonetheless, does play a role in the ribonucleotide reductase complex.     

Enzymatic regulation of the radical content of the small subunit of Escherichia coli ribonucleotide reductase involving reduction of its redox centers

The active form of protein B2, a homodimeric subunit of Escherichia coli ribonucleotide reductase, contains a diferric iron center and a cationic free radical localized to tyrosine 122 of one of the two polypeptide chains. Hydroxyurea scavenges this radical but leaves the iron center intact. The resulting metB2 (earlier named B2/HU) is enzymatically inactive. Crude extracts of E. coli catalyze the interconversion of metB2 and B2. Radical introduction into metB2 requires a flavin reductase together with a second poorly defined protein fraction ("Fraction b") as well as dioxygen, NAD(P)H, and a flavin (Fontecave, M., Eliasson, R., and Reichard, P. (1987) J. Biol. Chem. 262, 12325-12331). We now find that ferrous ions can substitute for Fraction b and that the diferric center of metB2 is reduced during anaerobic incubation of the system with reduced flavin and ferrous ions. Spectroscopic evidence and isotope experiments suggest an in situ reduction of the diferric to a diferrous center. Admission of oxygen then results in the instantaneous oxidation of tyrosine 122 to the cationic radical coupled to the reformation of the diferric center, giving enzymatically active B2. These data suggest that reduced diferrous B2 is an intermediate between metB2 and B2 during radical introduction. In addition, we find that anaerobic incubation of B2 with reduced flavin results in the loss of the tyrosyl radical and the formation of metB2. This reaction occurs in the absence of Fraction b or ferrous ions. Our experiments reconstitute with defined reagents the interconversion between metB2 and B2 observed earlier in the E. coli extract. The flavin reductase system catalyzes the interconversion in both directions with dioxygen as the critical factor deciding whether activation or inactivation of ribonucleotide reductase occurs.     

NAD(P)H:flavin oxidoreductase of Escherichia coli. A ferric iron reductase participating in the generation of the free radical of ribonucleotide reductase

The active form of one subunit of Escherichia coli ribonucleotide reductase (protein B2) contains an organic free radical localized to tyrosine 122 of its polypeptide chain. When this radical is scavenged, e.g. by treatment with hydroxyurea, the enzyme is inactivated (protein B2/HU). E. coli contains an enzyme system consisting of at least three proteins that in the presence of NADPH, FMN, dithiothreitol, and oxygen introduce the tyrosyl radical into B2/HU (Eliasson, R., J?rnvall, H., and Reichard, P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2373-2377). One of the three proteins was identified as superoxide dismutase. We now identify a second protein, previously provisionally named Fraction c, as an NAD(P)H:flavin oxidoreductase (flavin reductase). After 4,000-fold purification the protein moved as a single band on sodium dodecyl sulfate gel electrophoresis with a molecular weight of 28,000-29,000. The enzyme contained no flavin but reduced riboflavin, FMN, and FAD by NADH, or riboflavin and FMN by NADPH. It is a powerful ferric iron reductase. We propose that its complementing activity during radical generation involves participation in the reduction of the ferric iron center of protein B2/HU. Radical formation is then linked to the reoxidation of iron by oxygen. The flavin reductase may also participate in other aspects of iron metabolism of E. coli.     

A new tyrosyl radical on Phe208 as ligand to the diiron center in Escherichia coli ribonucleotide reductase, mutant R2-Y122H. Combined x-ray diffraction and EPR/ENDOR studies

The R2 protein subunit of class I ribonucleotide reductase (RNR) belongs to a structurally related family of oxygen bridged diiron proteins. In wild-type R2 of Escherichia coli, reductive cleavage of molecular oxygen by the diferrous iron center generates a radical on a nearby tyrosine residue (Tyr122), which is essential for the enzymatic activity of RNR, converting ribonucleotides into deoxyribonucleotides. In this work, we characterize the mutant E. coli protein R2-Y122H, where the radical site is substituted with a histidine residue. The x-ray structure verifies the mutation. R2-Y122H contains a novel stable paramagnetic center which we name H, and which we have previously proposed to be a diferric iron center with a strongly coupled radical, Fe(III)Fe(III)R.. Here we report a detailed characterization of center H, using 1H/2H -14N/15N- and 57Fe-ENDOR in comparison with the Fe(III)Fe(IV) intermediate X observed in the iron reconstitution reaction of R2. Specific deuterium labeling of phenylalanine residues reveals that the radical results from a phenylalanine. As Phe208 is the only phenylalanine in the ligand sphere of the iron site, and generation of a phenyl radical requires a very high oxidation potential, we propose that in Y122H residue Phe208 is hydroxylated, as observed earlier in another mutant (R2-Y122F/E238A), and further oxidized to a phenoxyl radical, which is coordinated to Fe1. This work demonstrates that small structural changes can redirect the reactivity of the diiron site, leading to oxygenation of a hydrocarbon, as observed in the structurally similar methane monoxygenase, and beyond, to formation of a stable iron-coordinated radical.     

Nitration of the tyrosyl radical in ribonucleotide reductase by nitrogen dioxide: a gamma radiolysis study

Nitrogen dioxide is a product of peroxynitrite homolysis and peroxidase-catalyzed oxidation of nitrite. It is of great importance in protein tyrosine nitration because most nitration pathways end with the addition of *NO2 to a one-electron-oxidized tyrosine. The rate constant of this radical addition reaction is high with free tyrosine-derived radicals. However, little is known of tyrosine radicals in proteins. In this paper, we have used *NO2 generated by gamma radiolysis to study the nitration of the R2 subunit of ribonucleotide reductase, which contains a long-lived tyrosyl radical on Tyr122. Most of the nitration occurred on Tyr122, but nonradical tyrosines were also modified. In addition, peptidic bonds close to nitrated Tyr122 could be broken. Nitration at Tyr122 was not observed with a radical-free metR2 protein. The estimated rate constant of the Tyr122 radical reaction with *NO2 was of 3 x 10(4) M(-1) s(-1), thus several orders of magnitude lower than that of a radical on free tyrosine. Nitration rate of other tyrosine residues in R2 was even lower, with an estimated value of 900 M(-1) s(-1). This study shows that protein environment can significantly reduce the reactivity of a tyrosyl radical. In ribonucleotide reductase, the catalytically active radical residue is very efficiently protected against nitrogen oxide attack and subsequent nitration.     

Activation of the iron-containing B2 protein of ribonucleotide reductase by hydrogen peroxide

The active form of protein B2, the small subunit of ribonucleotide reductase, contains two dinuclear Fe(III) centers and a tyrosyl radical. The inactive metB2 form also contains the same diferric complexes but lacks the tyrosyl radical. We now demonstrate that incubation of metB2 with hydrogen peroxide generates the tyrosyl radical. The reaction is optimal at 5.5 nM hydrogen peroxide, with a maximum of 25-30% tyrosyl radical being formed after approximately 1.5 hr of incubation. The activation reaction is counteracted by a hydrogen peroxide-dependent reduction of the tyrosyl radical. It is likely that the generation of the radical proceeds via a ferryl intermediate, as in the proposed mechanisms for cytochrome P-450 and the peroxidases.     

Paramagnetically shifted resonances in 1H NMR spectra of ribonucleotide reductase from Escherichia coli

The 400-MHz 1H NMR spectra of the subunit B2 of ribonucleotide reductase from Escherichia coli show paramagnetically shifted resonances at 24 ppm (exchangeable protons) and at 19 ppm (nonexchangeable protons). The protein contains an antiferromagnetically coupled dimeric iron center and a tyrosyl free radical. The paramagnetically shifted resonances must be due to the iron center, since they remain essentially unchanged in protein B2 with and without free radical. In analogy with recently published results for hemerythrin from Phascolopsis gouldii, which has a similar iron center, the 24-ppm resonance is suggested to arise from histidine ligands to the iron ions.     

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.     

Mutations in the R2 subunit of ribonucleotide reductase that confer resistance to hydroxyurea

Ribonucleotide reductase is an essential enzyme that catalyzes the reduction of ribonucleotides to deoxyribonucleotides for use in DNA synthesis. Ribonucleotide reductase from Escherichia coli consists of two subunits, R1 and R2. The R2 subunit contains an unusually stable radical at tyrosine 122 that participates in catalysis. Buried deep within a hydrophobic pocket, the radical is inaccessible to solvent although subject to inactivation by radical scavengers. One such scavenger, hydroxyurea, is a highly specific inhibitor of ribonucleotide reductase and therefore of DNA synthesis; thus it is an important anticancer and antiviral agent. The mechanism of radical access remains to be established; however, small molecules may be able to access Tyr-122 directly via channels from the surface of the protein. We used random oligonucleotide mutagenesis to create a library of 200,000 R2 mutants containing random substitutions at five contiguous residues (Ile-74, Ser-75, Asn-76, Leu-77, Lys-78) that partially comprise one side of a channel where Tyr-122 is visible from the protein surface. We subjected this library to increasing concentrations of hydroxyurea and identified mutants that enhance survival more than 1000-fold over wild-type R2 at high drug concentrations. Repetitive selections yielded S75T as the predominant R2 mutant in our library. Purified S75TR2 exhibits a radical half-life that is 50% greater than wild-type R2 in the presence of hydroxyurea. These data represent the first demonstration of R2 protein mutants in E. coli that are highly resistant to hydroxyurea; elucidation of their mechanism of resistance may provide valuable insight into the development of more effective inhibitors.     

Characterization of the free radical of mammalian ribonucleotide reductase

Mouse fibroblast 3T6 cells, selected for resistance to hydroxyurea, were shown to overproduce protein M2, one of the two nonidentical subunits of mammalian ribonucleotide reductase. Packed resistant cells gave an EPR signal at 77 K very much resembling the signal given by the tyrosine-free radical of the B2 subunit of Escherichia coli ribonucleotide reductase. Also, the M2-specific free radical was shown to be located at a tyrosine residue. Of the known tyrosine-free radicals of ribonucleotide reductases from E. coli, bacteriophage T4 infected E. coli and pseudorabies virus infected mouse L cells, the M2-specific EPR signal is most closely similar to the signal of the T4 radical. The small differences in the low temperature EPR signals between these four highly conserved tyrosine-free radical structures can be explained by slightly different angles of the beta-methylene group in relation to the plane of the aromatic ring of tyrosine, reflecting different conformations of the polypeptide chain around the tyrosines. The pronounced difference in microwave saturation between the E. coli B2 tyrosine radical EPR signal and the M2 signal could be due to their different interactions with unspecific paramagnetic ions or with the antiferromagnetically coupled iron pair, shown to be present in the E. coli enzyme and postulated also for the mammalian enzyme. A difference in the iron-radical center between the bacterial and mammalian ribonucleotide reductase is also observed in the ability to regenerate the free radical structure. In contrast to the B2 radical, the M2 tyrosine free radical could be regenerated by merely adding dithiothreitol in the presence of O2 to a cell extract where the radical had previously been destroyed by hydroxyurea treatment.     

The tyrosine free radical in ribonucleotide reductase from Escherichia coli.

One of the two nonidentical subunits of ribonucleotide reductase from Escherichia coli, protein B2, contains an organic free radical required for enzyme activity. Earlier isotope subtitution experiments (Sj?berg, B.-M., Reichard, P. Gr?slund, A., and Ehrenberg, A. (1977) J. Biol. Chem. 252, 536-541) demonstrated that the radical was localized to a tyrosine residue of the enzyme and suggested that the spin density of the radical was centered at the methylene carbon of tyrosine. However, additional isotope substitution experiments now show that the spin density of the radical must be delocalized over the aromatic ring of the tyrosine residue.     

Early loss of the tyrosyl radical in ribonucleotide reductase of adenocarcinoma cells producing nitric oxide.

Nitric oxide (NO) has been previously shown to inhibit crude preparations of ribonucleotide reductase, a key enzyme in DNA synthesis, and to destroy the essential tyrosyl free radical in pure recombinant R2 subunit of the enzyme. In R2-overexpressing TA3 cells, a decrease in the tyrosyl radical was observed by whole-cell EPR spectroscopy, as soon as 4 h after NO synthase induction by immunological stimuli. Complete loss of the tyrosyl EPR signal occurred after 7 h in cells cultured at a high density. Disappearance of the tyrosyl radical was prevented by N omega-nitro-L-arginine, a specific inhibitor of NO synthesis, and by oxyhemoglobin, which reacts rapidly with NO. It was reproduced by S-nitrosoglutathione, a NO-releasing molecule. Stable end products of NO synthase metabolism did not affect the radical. Immunoblot analysis of the R2 subunit indicated that expression of the protein was not influenced by NO synthase activity. These results establish that NO, or a labile product of NO synthase, induces the disappearance of the R2-centered tyrosyl radical. Since the radical is necessary for ribonucleotide reductase activity, its destruction by NO would contribute markedly to the antiproliferative action exerted by macrophage-type NO synthase.     

Structure and interactions of amino acid radicals in class I ribonucleotide reductase studied by ENDOR and high-field EPR spectroscopy

This short review compiles high-field electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) studies on different intermediate amino acid radicals, which emerge in wild-type and mutant class I ribonucleotide reductase (RNR) both in the reaction of protein subunit R2 with molecular oxygen, which generates the essential tyrosyl radical, and in the catalytic reaction, which involves a radical transfer between subunits R2 and R1. Recent examples are presented, how different amino acid radicals (tyrosyl, tryptophan, and different cysteine-based radicals) were identified, assigned to a specific residue, and their interactions, in particular hydrogen bonding, were investigated using high-field EPR and ENDOR spectroscopy. Thereby, unexpected diiron-radical centers, which emerge in mutants of R2 with changed iron coordination, and an important catalytic cysteine-based intermediate in the substrate turnover reaction in R1 were identified and characterized. Experiments on the essential tyrosyl radical in R2 single crystals revealed the so far unknown conformational changes induced by formation of the radical. Interesting structural differences between the tyrosyl radicals of class Ia and Ib enzymes were revealed. Recently accurate distances between the tyrosyl radicals in the protein dimer R2 could be determined using pulsed electron-electron double resonance (PELDOR), providing a new tool for docking studies of protein subunits. These studies show that high-field EPR and ENDOR are important tools for the identification and investigation of radical intermediates, which contributed significantly to the current understanding of the reaction mechanism of class I RNR.     

Structure and interactions of amino acid radicals in class I ribonucleotide reductase studied by ENDOR and high-field EPR spectroscopy

This short review compiles high-field electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) studies on different intermediate amino acid radicals, which emerge in wild-type and mutant class I ribonucleotide reductase (RNR) both in the reaction of protein subunit R2 with molecular oxygen, which generates the essential tyrosyl radical, and in the catalytic reaction, which involves a radical transfer between subunits R2 and R1. Recent examples are presented, how different amino acid radicals (tyrosyl, tryptophan, and different cysteine-based radicals) were identified, assigned to a specific residue, and their interactions, in particular hydrogen bonding, were investigated using high-field EPR and ENDOR spectroscopy. Thereby, unexpected diiron-radical centers, which emerge in mutants of R2 with changed iron coordination, and an important catalytic cysteine-based intermediate in the substrate turnover reaction in R1 were identified and characterized. Experiments on the essential tyrosyl radical in R2 single crystals revealed the so far unknown conformational changes induced by formation of the radical. Interesting structural differences between the tyrosyl radicals of class Ia and Ib enzymes were revealed. Recently accurate distances between the tyrosyl radicals in the protein dimer R2 could be determined using pulsed electron-electron double resonance (PELDOR), providing a new tool for docking studies of protein subunits. These studies show that high-field EPR and ENDOR are important tools for the identification and investigation of radical intermediates, which contributed significantly to the current understanding of the reaction mechanism of class I RNR.