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.     

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.     

The active form of the R2F protein of class Ib ribonucleotide reductase from Corynebacterium ammoniagenes is a diferric protein

Corynebacterium ammoniagenes contains a ribonucleotide reductase (RNR) of the class Ib type. The small subunit (R2F) of the enzyme has been proposed to contain a manganese center instead of the dinuclear iron center, which in other class I RNRs is adjacent to the essential tyrosyl radical. The nrdF gene of C. ammoniagenes, coding for the R2F component, was cloned in an inducible Escherichia coli expression vector and overproduced under three different conditions: in manganese-supplemented medium, in iron-supplemented medium, and in medium without addition of metal ions. A prominent typical tyrosyl radical EPR signal was observed in cells grown in rich medium. Iron-supplemented medium enhanced the amount of tyrosyl radical, whereas cells grown in manganese-supplemented medium had no such radical. In highly purified R2F protein, enzyme activity was found to correlate with tyrosyl radical content, which in turn correlated with iron content. Similar results were obtained for the R2F protein of Salmonella typhimurium class Ib RNR. The UV-visible spectrum of the C. ammoniagenes R2F radical has a sharp 408-nm band. Its EPR signal at g = 2.005 is identical to the signal of S. typhimurium R2F and has a doublet with a splitting of 0.9 millitesla (mT), with additional hyperfine splittings of 0.7 mT. According to X-band EPR at 77-95 K, the inactive manganese form of the C. ammoniagenes R2F has a coupled dinuclear Mn(II) center. Different attempts to chemically oxidize Mn-R2F showed no relation between oxidized manganese and tyrosyl radical formation. Collectively, these results demonstrate that enzymatically active C. ammoniagenes RNR is a generic class Ib enzyme, with a tyrosyl radical and a diferric metal cofactor.     

Studies of ribonucleotide reductase in crucian carp-an oxygen dependent enzyme in an anoxia tolerant vertebrate

The enzyme ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides, the precursors for DNA. RNR requires a thiyl radical to activate the substrate. In RNR of eukaryotes (class Ia RNR), this radical originates from a tyrosyl radical formed in reaction with oxygen (O(2)) and a ferrous di-iron center in RNR. The crucian carp (Carassius carassius) is one of very few vertebrates that can tolerate several months completely without oxygen (anoxia), a trait that enables this fish to survive under the ice in small ponds that become anoxic during the winter. Previous studies have found indications of cell division in this fish after 7 days of anoxia. This appears nearly impossible, as DNA synthesis requires the production of new deoxyribonucleotides and therefore active RNR. We have here characterized RNR in crucian carp, to search for adaptations to anoxia. We report the full-length sequences of two paralogs of each of the RNR subunits (R1i, R1ii, R2i, R2ii, p53R2i and p53R2ii), obtained by cloning and sequencing. The mRNA levels of these subunits were measured with quantitative PCR and were generally well maintained in hypoxia and anoxia in heart and brain. We also report maintained or increased mRNA levels of the cell division markers proliferating cell nuclear antigen (PCNA), brain derived neurotrophic factor (BDNF) and Ki67 in anoxic hearts and brains. Electron paramagnetic resonance (EPR) measurements on in vitro expressed crucian carp R2 and p53R2 proteins gave spectra similar to mammalian RNRs, including previously unpublished human and mouse p53R2 EPR spectra. However, the radicals in crucian carp RNR small subunits, especially in the p53R2ii subunit, were very stable at 0°C. A long half-life of the tyrosyl radical during wintertime anoxia could allow for continued cell division in crucian carp.     

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.     

Orientation of the tyrosyl radical in Salmonella typhimurium class Ib ribonucleotide reductase determined by high field EPR of R2F single crystals

The R2 protein of class I ribonucleotide reductase (RNR) generates and stores a tyrosyl radical, located next to a diferric iron center, which is essential for ribonucleotide reduction and thus DNA synthesis. X-ray structures of class Ia and Ib proteins from various organisms served as bases for detailed mechanistic suggestions. The active site tyrosine in R2F of class Ib RNR of Salmonella typhimurium is located at larger distance to the diiron site, and shows a different side chain orientation, as compared with the tyrosine in R2 of class Ia RNR from Escherichia coli.No structural information has been available for the active tyrosyl radical in R2F. Here we report on high field EPR experiments of single crystals of R2F from S. typhimurium, containing the radical Tyr-105*. Full rotational pattern of the spectra were recorded, and the orientation of the g-tensor axes were determined, which directly reflect the orientation of the radical Tyr-105* in the crystal frame. Comparison with the orientation of the reduced tyrosine Tyr-105-OH from the x-ray structure reveals a rotation of the tyrosyl side chain, which reduces the distance between the tyrosyl radical and the nearest iron ligands toward similar values as observed earlier for Tyr-122* in E. coli R2. Presence of the substrate binding subunit R1E did not change the EPR spectra of Tyr-105*, indicating that binding of R2E alone induces no structural change of the diiron site. The present study demonstrates that structural and functional information about active radical states can be obtained by combining x-ray and high-field-EPR crystallography.     

EPR evidence of two structurally different diferric sites in Mycobacterium tuberculosis R2-2 ribonucleotide reductase protein

Mixed-valent species were generated in the diiron site of active (with tyrosyl free radical) and met (without radical) forms of protein R2-2 in a class Ib ribonucleotide reductase from Mycobacterium tuberculosis by low temperature reduction (γ-irradiation) at 77 K. The primary mixed-valent EPR signal is a mixture of two components with axial symmetry and g_av<2.0, observable at temperatures up to 77 K, and assigned to antiferromagnetically coupled high spin ferric/ferrous sites. The two components in the primary EPR signal can be explained by the existence of two structurally distinct μ-oxo-bridged diferric centers, possibly related to structural heterogeneity around the iron site, and/or different properties of the two polypeptide chains in the homodimeric protein after the radical reconstitution reaction. Annealing of the irradiated R2-2 samples to 143 K transforms the primary EPR signal into a rhombic spectrum characterized by g_av<1.8 and observable only below 25 K. This spectrum is assigned to a partially relaxed form with a μ-hydroxo-bridge. Further annealing at 228 K produces a new complex rhombic EPR spectrum composed of at least two components. An identical EPR spectrum was observed and found to be stable upon chemical reduction of Mycobacterium tuberculosis RNR R2-2 at 293 K by dithionite.     

The iron-oxygen reconstitution reaction in protein R2-Tyr-177 mutants of mouse ribonucleotide reductase. Epr and electron nuclear double resonance studies on a new transient tryptophan radical.

The ferrous iron/oxygen reconstitution reaction in protein R2 of mouse and Escherichia coli ribonucleotide reductase (RNR) leads to the formation of a stable protein-linked tyrosyl radical and a mu-oxo-bridged diferric iron center, both necessary for enzyme activity. We have studied the reconstitution reaction in three protein R2 mutants Y177W, Y177F, and Y177C of mouse RNR to investigate if other residues at the site of the radical forming Tyr-177 can harbor free radicals. In Y177W we observed for the first time the formation of a tryptophan radical in protein R2 of mouse RNR with a lifetime of several minutes at room temperature. We assign it to an oxidized neutral tryptophan radical on Trp-177, based on selective deuteration and EPR and electron nuclear double resonance spectroscopy in H2O and D2O solution. The reconstitution reaction at 22 degrees C in both Y177F and Y177C leads to the formation of a so-called intermediate X which has previously been assigned to an oxo (hydroxo)-bridged Fe(III)/Fe(IV) cluster. Surprisingly, in both mutants that do not have successor radicals as Trp. in Y177W, this cluster exists on a much longer time scale (several seconds) at room temperature than has been reported for X in E. coli Y122F or native mouse protein R2. All three mouse R2 mutants were enzymatically inactive, indicating that only a tyrosyl radical at position 177 has the capability to take part in the reduction of substrates.     

Inhibition of chlamydial class Ic ribonucleotide reductase by C‐terminal peptides from protein R2

Chlamydia trachomatis ribonucleotide reductase (RNR) is a class Ic RNR. It has two homodimeric subunits: proteins R1 and R2. Class Ic protein R2 in its most active form has a manganese–iron metal cofactor, which functions in catalysis like the tyrosyl radical in classical class Ia and Ib RNRs. Oligopeptides with the same sequence as the C‐terminus of C. trachomatis protein R2 inhibit the catalytic activity of C. trachomatis RNR, showing that the class Ic enzyme shares a similar highly specific inhibition mechanism with the previously studied radical‐containing class Ia and Ib RNRs. The results indicate that the catalytic mechanism of this class of RNRs with a manganese–iron cofactor is similar to that of the tyrosyl‐radical‐containing RNRs, involving reversible long‐range radical transfer between proteins R1 and R2. The competitive binding of the inhibitory R2‐derived oligopeptide blocks the transfer pathway. We have constructed three‐dimensional structure models of C. trachomatis protein R1, based on homologous R1 crystal structures, and used them to discuss possible binding modes of the peptide to protein R1. Typical half maximal inhibitory concentration values for C. trachomatis RNR are about 200 µ m for a 20‐mer peptide, indicating a less efficient inhibition compared with those for an equally long peptide in the Escherichia coli class Ia RNR. A possible explanation is that the C. trachomatis R1/R2 complex has other important interactions, in addition to the binding mediated by the R1 interaction with the C‐terminus of protein R2. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

EPR evidence of two structurally different diferric sites in Mycobacterium tuberculosis R2-2 ribonucleotide reductase protein

Mixed-valent species were generated in the diiron site of active (with tyrosyl free radical) and met (without radical) forms of protein R2-2 in a class Ib ribonucleotide reductase from Mycobacterium tuberculosis by low temperature reduction (γ-irradiation) at 77 K. The primary mixed-valent EPR signal is a mixture of two components with axial symmetry and g_av<2.0, observable at temperatures up to 77 K, and assigned to antiferromagnetically coupled high spin ferric/ferrous sites. The two components in the primary EPR signal can be explained by the existence of two structurally distinct μ-oxo-bridged diferric centers, possibly related to structural heterogeneity around the iron site, and/or different properties of the two polypeptide chains in the homodimeric protein after the radical reconstitution reaction. Annealing of the irradiated R2-2 samples to 143 K transforms the primary EPR signal into a rhombic spectrum characterized by g_av<1.8 and observable only below 25 K. This spectrum is assigned to a partially relaxed form with a μ-hydroxo-bridge. Further annealing at 228 K produces a new complex rhombic EPR spectrum composed of at least two components. An identical EPR spectrum was observed and found to be stable upon chemical reduction of Mycobacterium tuberculosis RNR R2-2 at 293 K by dithionite.     

Spectroscopic and theoretical approaches for studying radical reactions in class I ribonucleotide reductase

Ribonucleotide reductases (RNRs) catalyze the production of deoxyribonucleotides, which are essential for DNA synthesis and repair in all organisms. The three currently known classes of RNRs are postulated to utilize a similar mechanism for ribonucleotide reduction via a transient thiyl radical, but they differ in the way this radical is generated. Class I RNR, found in all eukaryotic organisms and in some eubacteria and viruses, employs a diferric iron center and a stable tyrosyl radical in a second protein subunit, R2, to drive thiyl radical generation near the substrate binding site in subunit R1. From extensive experimental and theoretical research during the last decades, a general mechanistic model for class I RNR has emerged, showing three major mechanistic steps: generation of the tyrosyl radical by the diiron center in subunit R2, radical transfer to generate the proposed thiyl radical near the substrate bound in subunit R1, and finally catalytic reduction of the bound ribonucleotide. Amino acid- or substrate-derived radicals are involved in all three major reactions. This article summarizes the present mechanistic picture of class I RNR and highlights experimental and theoretical approaches that have contributed to our current understanding of this important class of radical enzymes.     

High catalytic activity achieved with a mixed manganese-iron site in protein R2 of Chlamydia ribonucleotide reductase

Ribonucleotide reductase (class I) contains two components: protein R1 binds the substrate, and protein R2 normally has a diferric site and a tyrosyl free radical needed for catalysis. In Chlamydia trachomatis RNR, protein R2 functions without radical. Enzyme activity studies show that in addition to a diiron cluster, a mixed manganese-iron cluster provides the oxidation equivalent needed to initiate catalysis. An EPR signal was observed from an antiferromagnetically coupled high-spin Mn(III)-Fe(III) cluster in a catalytic reaction mixture with added inhibitor hydroxyurea. The manganese-iron cluster in protein R2 confers much higher specific activity than the diiron cluster does to the enzyme.     

EPR studies on a stable sulfinyl radical observed in the iron-oxygen-reconstituted Y177F/I263C protein R2 double mutant of ribonucleotide reductase from mouse

Ribonucleotide reductase (RNR) catalyzes the biosynthesis of deoxyribonucleotides. The active enzyme contains a diiron center and a tyrosyl free radical required for enzyme activity. The radical is located at Y177 in the R2 protein of mouse RNR. The radical is formed concomitantly with the mu-oxo-bridged diferric center in a reconstitution reaction between ferrous iron and molecular oxygen in the protein. EPR at 9.6 and 285 GHz was used to investigate the reconstitution reaction in the double-mutant Y177F/I263C of mouse protein R2. The aim was to produce a protein-linked radical derived from the Cys residue in the mutant protein to investigate its formation and characteristics. The mutation Y177F hinders normal radical formation at Y177, and the I263C mutation places a Cys residue at the same distance from the iron center as Y177 in the native protein. In the reconstitution reaction, we observed small amounts of a transient radical with a probable assignment to a peroxy radical, followed by a stable sulfinyl radical, most likely located on C263. The unusual radical stability may be explained by the hydrophobic surroundings of C263, which resemble the hydrophobic pocket surrounding Y177 in native protein R2. The observation of a sulfinyl radical in RNR strengthens the relationship between RNR and another free radical enzyme, pyruvate formate-lyase, where a similar relatively stable sulfinyl radical has been observed in a mutant. Sulfinyl radicals may possibly be considered as stabilized forms of very short-lived thiyl radicals, proposed to be important intermediates in the radical chemistry of RNR.     

Electron paramagnetic resonance (EPR) spectroscopy of the stable-free radical in the native metallo-cofactor of the manganese-ribonucleotide reductase (Mn-RNR) of Corynebacterium glutamicum

Ribonucleotide reductases (RNR; EC 1.17.4.1) provide the 2′-deoxyribonucleotides for DNA replication of proliferating cells by a uniform radical mechanism using diverse metals. The native metallo-cofactor of the Corynebacterium glutamicum RNR contains manganese and is sensitive to EDTA and radical scavengers. Hybrid holoenzymes, capable of ribonucleotide reduction, were composed of the small manganese-containing (R2F) and the large catalytic subunit (R1E) from either of the two corynebacterial RNRs. A synthetic peptide deduced from the C-terminal region of the nrdF gene inhibited the C. glutamicum-RNR non-competitively and cross-reacted with the C. ammoniagenes-RNR. The C. glutamicum-R2F has a saturable organic radical signal at g=2.005 detected by electron paramagnetic resonance (EPR) spectroscopy and shows a distinct absorption at 408 nm indicative of a tyrosyl-like organic radical (Y·). Quantification of the metal content revealed 0.06 mol Fe but 0.8 mol Mn per mol R2F-monomer and would thus assign two manganese atoms bound to the dimeric metallo-cofactor, while a distinct enzymatic activity (32 µmol×mg^?1×min^?1) was observed in the biochemical complementation assay. Divergence of the C. glutamicum-RNR studied here from the prototypical Salmonella typhimurium class 1b enzyme and the Chlamydia trachomatis class Ic enzyme is discussed below.     

Investigation of in vivo diferric tyrosyl radical formation in Saccharomyces cerevisiae Rnr2 protein: requirement of Rnr4 and contribution of Grx3/4 AND Dre2 proteins

The β₂ subunit of class Ia ribonucleotide reductase (RNR) contains a diferric tyrosyl radical cofactor (Fe₂^III-Tyr^•) that is essential for nucleotide reduction. The β₂ subunit of Saccharomyces cerevisiae is a heterodimer of Rnr2 (β) and Rnr4 (β′). Although only β is capable of iron binding and Tyr^• formation, cells lacking β′ are either dead or exhibit extremely low Tyr^• levels and RNR activity depending on genetic backgrounds. Here, we present evidence supporting the model that β′ is required for iron loading and Tyr^• formation in β in vivo via a pathway that is likely dependent on the cytosolic monothiol glutaredoxins Grx3/Grx4 and the Fe-S cluster protein Dre2. rnr4 mutants are defective in iron loading into nascent β and are hypersensitive to iron depletion and the Tyr^•-reducing agent hydroxyurea. Transient induction of β′ in a GalRNR4 strain leads to a concomitant increase in iron loading and Tyr^• levels in β. Tyr^• can also be rapidly generated using endogenous iron when permeabilized Δrnr4 spheroplasts are supplemented with recombinant β′ and is inhibited by adding an iron chelator prior to, but not after, β′ supplementation. The growth defects of rnr4 mutants are enhanced by deficiencies in grx3/grx4 and dre2. Moreover, depletion of Dre2 in GalDRE2 cells leads to a decrease in both Tyr^• levels and ββ′ activity. This result, in combination with previous findings that a low level of Grx3/4 impairs RNR function, strongly suggests that Grx3/4 and Dre2 serve in the assembly of the deferric Tyr^• cofactor in RNR.     

Redox-induced structural changes in the di-iron and di-manganese forms of Bacillus anthracis ribonucleotide reductase subunit NrdF suggest a mechanism for gating of radical access

Class Ib ribonucleotide reductases (RNR) utilize a di-nuclear manganese or iron cofactor for reduction of superoxide or molecular oxygen, respectively. This generates a stable tyrosyl radical (Y·) in the R2 subunit (NrdF), which is further used for ribonucleotide reduction in the R1 subunit of RNR. Here, we report high-resolution crystal structures of Bacillus anthracis NrdF in the metal-free form (1.51 Å) and in complex with manganese (Mn^II/Mn^II, 1.30 Å). We also report three structures of the protein in complex with iron, either prepared anaerobically (Fe^II/Fe^II form, 1.32 Å), or prepared aerobically in the photo-reduced Fe^II/Fe^II form (1.63 Å) and with the partially oxidized metallo-cofactor (1.46 Å). The structures reveal significant conformational dynamics, likely to be associated with the generation, stabilization, and transfer of the radical to the R1 subunit. Based on observed redox-dependent structural changes, we propose that the passage for the superoxide, linking the FMN cofactor of NrdI and the metal site in NrdF, is closed upon metal oxidation, blocking access to the metal and radical sites. In addition, we describe the structural mechanics likely to be involved in this process.

     

Iron ligand mutants in protein R2 of Escherichia coli ribonucleotide reductase

 Ribonucleotide reductase protein R2 contains a diiron-oxo center with the ability to generate and stabilize a catalytically essential tyrosyl radical. The six protein-derived ligands (four carboxylates and two histidines) of the diiron site were, in separate experiments, mutated to alanines and in two cases also to histidines. We found that removal or exchange of an iron ligand did not in general abolish the formation of a diiron site in the mutant proteins, although all mutant proteins lost the bound metal ions with time upon storage. Iron bound to the mutant proteins was characterized by light absorption, EPR and resonance Raman spectroscopy. In addition, the ability of the mutant proteins to form a tyrosyl free radical and the catalytic competence of the latter were determined by EPR spectroscopy and activity measurements. The diiron sites of mutant proteins D84H and E238A were quite reminiscent of that in wild-type R2. Four of the other mutant proteins (H118A, E204A, E204H, H241A) could form the same number of metal sites as wild-type R2, but with different spectroscopic properties. The mutation E115A affecting the only μ-bridging ligand lowered the amount of bound iron to less than half. An important observation was that D84A, H118A and E204A formed transient tyrosyl radicals, but only the E204A mutant protein was enzymatically active. D84A and H118A affect iron ligands which have been suggested to participate in long-range electron transfer during catalysis. Our observation that these mutant proteins are catalytically inert, despite formation of a tyrosyl radical, underscores the necessity for an intact electron transfer pathway for catalytic activity in ribonucleotide reductase. Received: 31 August 1995 / Accepted: 14 February 1996     

A docking model of human ribonucleotide reductase with flavin and phenosafranine

Ribonucleotide Reductase (RNR) is an enzyme responsible for the reduction of ribonucleotides to their corresponding Deoxyribonucleotides (DNA), which is a building block for DNA replication and repair mechanisms. The key role of RNR in DNA synthesis and control in cell growth has made this an important target for anticancer therapy. Increased RNR activity has been associated with malignant transformation and tumor cell growth. In recent years, several RNR inhibitors, including Triapine, Gemcitabine and GTI-2040, have entered the clinical trials. Our current work focuses on an attempted to dock this inhibitors Flavin and Phenosafranine to curtail the action of human RNR2. The docked inhibitor Flavin and Phenosafranine binds at the active site with THR176, which are essential for free radical formation. The inhibitor must be a radical scavenger to destroy the tyrosyl radical or iron metal scavenger. The iron or radical site of R2 protein can react with one-electron reductants, whereby the tyrosyl radical is converted to a normal tyrosine residue. However, compounds such as Flavin and Phenosafranine were used in most of the cases to reduce the radical activity. The docking study was performed for the crystal structure of human RNR with the radical scavengers Flavin and Phenosafranine to inhibit the human RNR2. This helps to understand the functional aspects and also aids in the development of novel inhibitors for the human RNR2.     

Ribonucleotide reductase inhibition by p-alkoxyphenols studied by molecular docking and molecular dynamics simulations

Ribonucleotide reductase (RNR) is necessary for production of the precursor deoxyribonucleotides for DNA synthesis. Class Ia RNR functions via a stable free radical in one of the two components protein R2. The enzyme mechanism involves long range (proton coupled) electron transfer between protein R1 and the tyrosyl radical in protein R2. Earlier experimental studies showed that p-alkoxyphenols inhibit RNR. Here, molecular docking and molecular dynamics simulations involving protein R2 suggest an inhibition mechanism for p-alkoxyphenols . A low energy binding pocket is identified in protein R2. The preferred configuration provides a structural basis explaining their specific binding to the Escherichia coli and mouse R2 proteins. Trp48 (E. coli numbering), on the electron transfer pathway, is involved in the interactions with the inhibitors. The relative order of the binding energies calculated for the phenol derivatives to protein R2 is correlated with earlier experimental data on inhibition efficiency, in turn related to increasing size of the hydrophobic alkyl substituents. Using the configuration identified by molecular docking as a starting point for molecular dynamics simulations, we find that the p-allyloxyphenol interrupts the catalytic electron transfer pathway of the R2 protein by forming hydrogen bonds with Trp48 and Asp237, thus explaining the inhibitory activity of p-alkoxyphenols.