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.     

Compounds with capacity to quench the tyrosyl radical in Pseudomonas aeruginosa ribonucleotide reductase

JBIC Journal of Biological Inorganic Chemistry - Ribonucleotide reductase (RNR) has been extensively probed as a target enzyme in the search for selective antibiotics. Here we report on the...     

Loss of the tyrosyl radical in mouse ribonucleotide reductase by (-)-epicatechin

The flavonoid (-)-epicatechin was previously demonstrated to interfere with tyrosine nitration by peroxynitrite [Biochem. Biophys. Res. Commun. 285 (2001) 782]. This effect was hypothesized to be based upon an interaction of epicatechin with a transiently generated tyrosyl radical. In the present study, using electron paramagnetic resonance, we demonstrate that (-)-epicatechin is capable of destabilizing the tyrosyl radical of the mouse ribonucleotide reductase R2 component. First-order rate constants for the disappearance of tyrosyl radical signals were 1 x 10(-4) and 2 x 10(-4)s(-1)for epicatechin and hydroxyurea, a well-known tyrosyl radical scavenger, respectively. In keeping with scavenging the ribonucleotide reductase tyrosyl radical, cellular production of deoxyribonucleotides and DNA synthesis were impaired by (-)-epicatechin in normal human keratinocytes and in human squamous carcinoma cells.     

A stable FeIII-FeIV replacement of tyrosyl radical in a class I ribonucleotide reductase

Ribonucleotide reductase (RNR) of Chlamydia trachomatis is a class I RNR enzyme composed of two homodimeric components, proteins R1 and R2. In class I RNR, R1 has the substrate binding site, whereas R2 has a diferric site and normally in its active form a stable tyrosyl free radical. C. trachomatis RNR is unusual, because its R2 component has a phenylalanine in the place of the radical carrier tyrosine. Replacing the tyrosyl radical, a paramagnetic Fe(III)-Fe(IV) species (species X, normally a transient intermediate in the process leading to radical formation) may provide the oxidation equivalent needed to start the catalytic process via long range electron transfer from the active site in R1. Here EPR spectroscopy shows that in C. trachomatis RNR, species X can become essentially stable when formed in a complete RNR (R1/R2/substrate) complex, adding further weight to the possible role of this species X in the catalytic reaction.     

Allosteric regulation of the class III anaerobic ribonucleotide reductase from bacteriophage T4

Ribonucleotide reductase (RNR) is an essential enzyme in all organisms. It provides precursors for DNA synthesis by reducing all four ribonucleotides to deoxyribonucleotides. The overall activity and the substrate specificity of RNR are allosterically regulated by deoxyribonucleoside triphosphates and ATP, thereby providing balanced dNTP pools. We have characterized the allosteric regulation of the class III RNR from bacteriophage T4. Our results show that the T4 enzyme has a single type of allosteric site to which dGTP, dTTP, dATP, and ATP bind competitively. The dissociation constants are in the micromolar range, except for ATP, which has a dissociation constant in the millimolar range. ATP and dATP are positive effectors for CTP reduction, dGTP is a positive effector for ATP reduction, and dTTP is a positive effector for GTP reduction. dATP is not a general negative allosteric effector. These effects are similar to the allosteric regulation of class Ib and class II RNRs, and to the class Ia RNR of bacteriophage T4, but differ from that of the class III RNRs from the host bacterium Escherichia coli and from Lactococcus lactis. The relative rate of reduction of the four substrates was measured simultaneously in a mixed-substrate assay, which mimics the physiological situation and illustrates the interplay between the different effectors in vivo. Surprisingly, we did not observe any significant UTP reduction under the conditions used. Balancing of the pyrimidine deoxyribonucleotide pools may be achieved via the dCMP deaminase and dCMP hydroxymethylase pathways.     

Structure and function of the radical enzyme ribonucleotide reductase

Ribonucleotide reductases (RNRs) catalyze all new production in nature of deoxyribonucleotides for DNA synthesis by reducing the corresponding ribonucleotides. The reaction involves the action of a radical that is produced differently for different classes of the enzyme. Class I enzymes, which are present in eukaryotes and microorganisms, use an iron center to produce a stable tyrosyl radical that is stored in one of the subunits of the enzyme. The other classes are only present in microorganisms. Class II enzymes use cobalamin for radical generation and class III enzymes, which are found only in anaerobic organisms, use a glycyl radical. The reductase activity is in all three classes contained in enzyme subunits that have similar structures containing active site cysteines. The initiation of the reaction by removal of the 3′-hydrogen of the ribose by a transient cysteinyl radical is a common feature of the different classes of RNR. This cysteine is in all RNRs located on the tip of a finger loop inserted into the center of a special barrel structure. A wealth of structural and functional information on the class I and class III enzymes can now give detailed views on how these enzymes perform their task. The class I enzymes demonstrate a sophisticated pattern as to how the free radical is used in the reaction, in that it is only delivered to the active site at exactly the right moment. RNRs are also allosterically regulated, for which the structural molecular background is now starting to be revealed.     

Magnetic interaction between the tyrosyl free radical and the antiferromagnetically coupled iron center in ribonucleotide reductase

Ribonucleotide reductases from Escherichia coli and from mammalian cells are heterodimeric enzymes. One of the subunits, in the bacterial enzyme protein B2 and in the mammalian enzyme protein M2, contains iron and a tyrosyl free radical that both are essential for enzyme activity. The iron center in protein B2 is an antiferromagnetically coupled pair of high-spin ferric ions. This study concerns magnetic interaction between the tyrosyl radical and the iron center in the two proteins. Studies of the temperature dependence of electron paramagnetic resonance (EPR) relaxation and line shape reveal significant differences between the free radicals in proteins B2 and M2. The observed temperature-dependent enhanced EPR relaxation and line broadening of the enzyme radicals are furthermore completely different from those of a model UV-induced free radical in tyrosine. The results are discussed in terms of magnetic dipolar as well as exchange interactions between the free radical and the iron center in both proteins. The free radical and the iron center are thus close enough in space to exhibit magnetic interaction. For protein M2 the effects are more pronounced than for protein B2, indicating a stronger magnetic interaction.     

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.     

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.     

The bacteriophage T4 gene for the small subunit of ribonucleotide reductase contains an intron.

The bacteriophage T4 gene nrdB codes for the small subunit of the enzyme ribonucleotide reductase. The T4 nrdB gene was localized between 136.1 kb and 137.8 kb in the T4 genetic map according to the deduced structural homology of the protein to the amino acid sequence of its bacterial counterpart, the B2 subunit of Escherichia coli. This positions the C-terminal end of the T4 nrdB gene approximately 2 kb closer to the T4 gene 63 than earlier anticipated from genetic recombinational analyses. The most surprising feature of the T4 nrdB gene is the presence of an approximately 625 bp intron which divides the structural gene into two parts. This is the second example of a prokaryotic structural gene with an intron. The first prokaryotic intron was reported in the nearby td gene, coding for the bacteriophage T4-specific thymidylate synthase enzyme. The nucleotide sequence at the exon-intron junctions of the T4 nrdB gene is similar to that of the junctions of the T4 td gene: the anticipated exon-intron boundary at the donor site ends with a TAA stop codon and there is an ATG start codon at the putative downstream intron-exon boundary of the acceptor site. In the course of this work the denA gene of T4 (endonuclease II) was also located.     

Cysteines involved in radical generation and catalysis of class III anaerobic ribonucleotide reductase. A protein engineering study of bacteriophage T4 NrdD

Class III ribonucleotide reductase (RNR) is an anaerobic glycyl radical enzyme that catalyzes the reduction of ribonucleotides to deoxyribonucleotides. We have investigated the importance in the reaction mechanism of nine conserved cysteine residues in class III RNR from bacteriophage T4. By using site-directed mutagenesis, we show that two of the cysteines, Cys-79 and Cys-290, are directly involved in the reaction mechanism. Based on the positioning of these two residues in the active site region of the known three-dimensional structure of the phage T4 enzyme, and their structural equivalence to two cysteine residues in the active site region of the aerobic class I RNR, we suggest that Cys-290 participates in the reaction mechanism by forming a transient thiyl radical and that Cys-79 participates in the actual reduction of the substrate. Our results provide strong experimental evidence for a similar radical-based reaction mechanism in all classes of RNR but also identify important differences between class III RNR and the other classes of RNR as regards the reduction per se. We also identify a cluster of four cysteines (Cys-543, Cys-546, Cys-561, and Cys-564) in the C-terminal part of the class III enzyme, which are essential for formation of the glycyl radical. These cysteines make up a CX(2)C-CX(2)C motif in the vicinity of the stable radical at Gly-580. We propose that the four cysteines are involved in radical transfer between Gly-580 and the cofactor S-adenosylmethionine of the activating NrdG enzyme needed for glycyl radical generation.     

Spectroscopic studies of the iron and manganese reconstituted tyrosyl radical in Bacillus cereus ribonucleotide reductase R2 protein

Ribonucleotide reductase (RNR) catalyzes the rate limiting step in DNA synthesis where ribonucleotides are reduced to the corresponding deoxyribonucleotides. Class Ib RNRs consist of two homodimeric subunits: R1E, which houses the active site; and R2F, which contains a metallo cofactor and a tyrosyl radical that initiates the ribonucleotide reduction reaction. We studied the R2F subunit of B. cereus reconstituted with iron or alternatively with manganese ions, then subsequently reacted with molecular oxygen to generate two tyrosyl-radicals. The two similar X-band EPR spectra did not change significantly over 4 to 50 K. From the 285 GHz EPR spectrum of the iron form, a g ₁-value of 2.0090 for the tyrosyl radical was extracted. This g ₁-value is similar to that observed in class Ia E. coli R2 and class Ib R2Fs with iron-oxygen cluster, suggesting the absence of hydrogen bond to the phenoxyl group. This was confirmed by resonance Raman spectroscopy, where the stretching vibration associated to the radical (C-O, ν_7a = 1500 cm⁻¹) was found to be insensitive to deuterium-oxide exchange. Additionally, the ¹⁸O-sensitive Fe-O-Fe symmetric stretching (483 cm⁻¹) of the metallo-cofactor was also insensitive to deuterium-oxide exchange indicating no hydrogen bonding to the di-iron-oxygen cluster, and thus, different from mouse R2 with a hydrogen bonded cluster. The HF-EPR spectrum of the manganese reconstituted RNR R2F gave a g ₁-value of ∼2.0094. The tyrosyl radical microwave power saturation behavior of the iron-oxygen cluster form was as observed in class Ia R2, with diamagnetic di-ferric cluster ground state, while the properties of the manganese reconstituted form indicated a magnetic ground state of the manganese-cluster. The recent activity measurements (Crona et al., (2011) J Biol Chem 286: 33053–33060) indicates that both the manganese and iron reconstituted RNR R2F could be functional. The manganese form might be very important, as it has 8 times higher activity.     

Effect of hydroxyurea on T4 ribonucleotide reductase.

Phage T4-induced ribonucleotide reductase, purified to homogeneity, catalyzes the reduction of the four ribonucleotides CDP, UDP, ADP, and GDP to the corresponding deoxyribonucleotides. The enzyme is an order of magnitude more sensitive to hydroxyurea than the corresponding Escherichia coli enzyme. Fifty per cent inhibition occurs at 10 micrometer hydroxyurea. Inhibition is complete at a high concentration of the drug, and there is no differential effect on the four substrates. Treatment of T4 ribonucleotide reductase or its isolated subunits with hydroxyurea does not lead to their irreversible inactivation.     

EPR stopped-flow studies of the reaction of the tyrosyl radical of protein R2 from ribonucleotide reductase with hydroxyurea.

The reaction of the functional tyrosyl radical in protein R2 of ribonucleotide reductase from E. coli and mouse with the enzyme inhibitor hydroxyurea has been studied by EPR stopped-flow techniques at room temperature. The rate of disappearance of the tyrosyl radical in E. coli protein R2 is k2 = 0.43 M-1 s-1 at 25 degrees C. The reaction follows pseudo-first-order kinetics up to 450 mM hydroxyurea indicating that no saturation by hydroxyurea takes place even at this high concentration. Transient nitroxide-like radicals from hydroxyurea have been detected for the first time in the reaction of hydroxyurea with protein R2 from E. coli and mouse, indicating that 1-electron transfer from hydroxyurea to the tyrosyl radical is the dominating mechanism in the inhibitor reaction. The hydroxyurea radicals appear in low steady-state concentrations during 2-3 half-decay times of the tyrosyl radical and disappear thereafter.     

Resonance Raman spectroscopy of ribonucleotide reductase. Evidence for a deprotonated tyrosyl radical and photochemistry of the binuclear iron center

Native ribonucleotide reductase from Escherichia coli exhibits a resonance-enhanced Raman mode at 1498 cm-1 that is characteristic of a tyrosyl radical. The Raman frequency as well as the absorption maximum at 410 nm identifies the radical as being in a deprotonated state. The B2 subunit of ribonucleotide reductase shows an additional resonance Raman mode at 493 cm-1 that has been assigned to the symmetric stretch of an Fe-O-Fe moiety. When samples of active B2 or metB2 are exposed to a tightly focused laser beam at 406.7 nm, there is a loss of intensity at 493 cm-1 and the appearance of a new peak at 595 cm-1. Although the 595-cm-1 feature was previously assigned to an Fe-OH vibration on the basis of its 23-cm-1 shift to lower energy in H2(18)O and the apparent dependence of its intensity on pH [Sj?berg, B. M., Loehr, T. M., & Sanders-Loehr, J. (1987) Biochemistry 26, 4242], the present studies indicate that the intensity of this mode is dependent primarily on input laser power. The peak at 595 cm-1 is more plausibly assigned to a new vs(Fe-O-Fe) mode in view of its lack of the deuterium isotope dependence expected for an Fe-OH mode and its resonant scattering cross section which is comparable to that of the 493-cm-1 mode. This new species has a calculated Fe-O-Fe angle of approximately 113 degrees compared to approximately 138 degrees calculated for the Fe-O-Fe unit in unmodified protein B2. One possible explanation for the photoinduced vibrational mode is that a bridging solvent molecule has been inserted in place of a bridging carboxylate.     

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.     

Two active site asparagines are essential for the reaction mechanism of the class III anaerobic ribonucleotide reductase from bacteriophage T4

Class III ribonucleotide reductase is an anaerobic enzyme that uses a glycyl radical to catalyze the reduction of ribonucleotides to deoxyribonucleotides and formate as ultimate reductant. The reaction mechanism of class III ribonucleotide reductases requires two cysteines within the active site, Cys-79 and Cys-290 in bacteriophage T4 NrdD numbering. Cys-290 is believed to form a transient thiyl radical that initiates the reaction with substrate and Cys-79 to take part as a transient thiyl radical in later steps of the reductive reaction. The recently solved three-dimensional structure of class III ribonucleotide reductase (RNR) from bacteriophage T4 shows that two highly conserved asparagines, Asn-78 and Asn-311, are positioned close to the essential Cys-79. We have investigated the function of Asn-78 and Asn-311 by site-directed mutagenesis and measured enzyme activity and glycyl radical formation in five single (N78(A/C/D) and N311(A/C)) and one double (N78A/N311A) mutant proteins. Our results suggest that both asparagines are important for the catalytic mechanism of class III RNR and that one asparagine can partially compensate for the lack of the other functional group in the single Asn --> Ala mutant proteins. A plausible role for these two asparagines could be in positioning formate in the active site to orient it toward the proposed thiyl radical of Cys-79. This would also control the highly reactive carbon dioxide radical anion form of formate within the active site before it is released as carbon dioxide. A detailed reaction scheme including the function of the two asparagines and two formate molecules is proposed for class III RNRs.