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Bacillus subtilis class Ib ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, providing the building blocks for DNA replication and repair. It is composed of two proteins: α (NrdE) and β (NrdF). β contains the metallo-cofactor, essential for the initiation of the reduction process. The RNR genes are organized within the nrdI-nrdE-nrdF-ymaB operon. Each protein has been cloned, expressed, and purified from Escherichia coli. As isolated, recombinant NrdF (rNrdF) contained a diferric-tyrosyl radical [Fe(III)2-Y•] cofactor. Alternatively, this cluster could be self-assembled from apo-rNrdF, Fe(II), and O2. Apo-rNrdF loaded using 4 Mn(II)/β2, O2, and reduced NrdI (a flavodoxin) can form a dimanganese(III)-Y• [Mn(III)2-Y•] cofactor. In the presence of rNrdE, ATP, and CDP, Mn(III)2-Y• and Fe(III)2-Y• rNrdF generate dCDP at rates of 132 and 10 nmol min-1 mg-1, respectively (both normalized for 1 Y•/β2). To determine the endogenous cofactor of NrdF in B. subtilis, the entire operon was placed behind a Pspank(hy) promoter and integrated into the B. subtilis genome at the amyE site. All four genes were induced in cells grown in Luria-Bertani medium, with levels of NrdE and NrdF elevated 35-fold relative to that of the wild-type strain. NrdE and NrdF were copurified in a 1:1 ratio from this engineered B. subtilis. The visible, EPR, and atomic absorption spectra of the purified NrdENrdF complex (eNrdF) exhibited characteristics of a Mn(III)2-Y• center with 2 Mn/β2 and 0.5 Y•/β2 and an activity of 318-363 nmol min-1 mg-1 (normalized for 1 Y•/β2). These data strongly suggest that the B. subtilis class Ib RNR is a Mn(III)2-Y• enzyme. [ABSTRACT FROM AUTHOR]

Human ribonucleotide reductases (hRNRs) catalyze the conversion of nucleotides to deoxynucleotides and are composed of α- and β-subunits that form active anhim (n, m = 2 or 6) complexes. a binds NDP substrates (CDP, UDP, ADP, and GDP, C site) as well as ATP and dNTPs (dATP, dGTP, TTP) allosteric effectors that control enzyme activity (A site) and substrate specificity (S site). Clofarabine (ClF), an adenosine analog, is used in the treatment of refractory leukemias. Its mode of cytotoxicity is thought to be associated in part with the triphosphate functioning as an allosteric inhibitor of hRNR. Studies on the mechanism of inhibition of hRNR by ClF di- and triphosphates (ClFDP and ClFTP) are presented. ClFTP is a reversible inhibitor (Ki = 40 nM) that rapidly inactivates hRNR. However, with time, 50% of the activity is recovered. D57N-α, a mutant with an altered A site, prevents inhibition by ClFTP, suggesting its A site binding. ClFDP is a slow-binding, reversible inhibitor (Ki = 17 nM; t1/2 = 23 min). CDP protects a from its inhibition. The altered off-rate of ClFDP from E • ClFDP' by ClFTP (A site) or dGTP (S site) and its inhibition of D57N-α together implicate its (site binding. Size exclusion chromatography of hRNR or a alone with ClFDP or ClFTP, ±ATP or dGTP, reveals in each case that a forms a kinetically stable hexameric state. This is the first example of hexamerization of a induced by an NDP analog that reversibly binds at the active site. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductase (RNR) from Lactobacillus leichmannii, a 76 kDa monomer using adenosyleobalamin (AdoCbl) as a cofactor, catalyzes the conversion of nucleoside triphosphates to deoxynuclèotides and is rapidly (<30 s) inactivated by I equiv of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F2CTP). [l-3H]. and [5-3H]F2CTP were synthesized and used independently to inactivate RNR. Sephadex G-50 chromatography of the inactivation mixture revealed that 0.47 equiv of a sugar was covalently bound to RN Rand that 0.71 equiv of eytosine was released. Alternatively, analysis of the inactivated RNR by SDS-PAGE without boiling resulted in 33% of RNR migrating as a 110 kDa protein. Inactivation of RNR with a mixlure of [I-3H]F2CTPand [l-2HIF2CTP followed by reduction with NaBH4, alkylation with iodoaeetamide, trypsin digestion, and HPLC separation of the resulting peptides allowed isolation and identification by MALDI-TOF mass speetrometry (MS) of a 3H/2H-labeled peptide containing C731 and C736 from the C-terminus of RNR accounting for 10% of the labeled protein. The MS analysis also revealed that the two eysteines were cross-linked to a furanone species derived from the sugar of F2CTP. Incubation of [l'-3H]F2CTP with C119S-RNR resulted in 0.3 equiv of sugar being covalently bound to the protein, and incubation with NaBH4 subsequent to inactivation resulted in trapping of 2'-fluoro-2'-deoxycytidine. These sludieS and the ones in the preceding paper (DOI: l0.1021/bi9021318) allow proposal of a mechanism of inactivation of RN R by F2CTP involving multiple reaction pathways. The proposed mechanisms share many common features with F2CDP inactivation of the class I RNRs. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides and is essential in all organisms. Class I RNRs consist of two homodimeric subunits: α2 and β2. The α subunit contains the site of nucleotide reduction, and the β subunit contains the essential diferric-tyrosyl radical (Y•) cofactor. Escherichia coli contains genes encoding two class I RNRs (Ia and Ib) and a class III RNR, which is active only under anaerobic conditions. Its class Ia RNR, composed of NrdA (α) and NrdB (β), is expressed under normal aerobic growth conditions. The class lb RNR, composed of NrdE (α) and NrdF (β), is expressed under oxidative stress and iron-limited growth conditions. Our laboratory is interested in pathways of cofactor biosynthesis and maintenance in class I RNRs and modulation of Y• levels as a means of regulating RNR activity. Our recent studies have implicated a [2Fe2S]-ferredoxin, YfaE, in the NrdB diferric-Y• maintenance pathway and possibly in the biosynthetic and regulatory pathways. Here, we report that NrdI is a flavodoxin counterpart to YfaE for the class lb RNR. It possesses redox properties unprecedented for a flavodoxin (Eox/sq = 264 ± 17 mV and Esq/hq = -255 ± 17 my) that allow it to mediate a two-electron reduction of the diferric cluster of NrdF via two successive one-electron transfers. Data presented support the presence of a distinct maintenance pathway for NrdEF, orthogonal to that for NrdAB involving YfaE. [ABSTRACT FROM AUTHOR]

New insights have been gained into the formation of the di-iron tyrosyl radical cofactor, which is essential for nucleotide reduction in the class I ribonucleotide reductases. Recent advances include Insight into the function of the tyrosyl radical in initiation of nucleotide reduction. [Copyright &y& Elsevier]

Presents a study on the description of the successful efforts in providing evidence that the nitrogen centered radical is covalently bound to the sulfur of a cysteine of the R1 subunit of E. coli ribonucleotide reductase (RNR) using electron paramagnetic resonance (EPR) spectroscopy together with RNR containing [beta-2H]cysteine.

Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleoside 5'-diphosphates (NDPs) to deoxynucleotides (dNDPs). The active site for NDP reduction resides in α2, and the essential diferric-tyrosyl radical (Y 122*) cofactor that initiates transfer of the radical to the active site cysteine in α2 (C439), 35 A removed, is in $2. The oxidation is proposed to involve a hopping mechanism through aromatic amino acids (Y122 W4s -. Y356 in $2 to Y731 → Y730 → C439 in α2) and reversible proton- coupled electron transfer (PCET). Recently, 2,3,5-F3Y (F3Y) was site-specifically incorporated in place of Y356 in α2 and 3-NH2Y (NH2Y) in place of Y731 and Y730 in α2. A pH-rate profile with F3Y356-β2 suggested that as the pH is elevated, the rate-determining step of RNR can be altered from a conformational change to PCET and that the altered driving force for F3Y oxidation, by residues adjacent to it in the pathway, is responsible for this change. Studies with NH2Y731(730>-a2, α2, CDP, and ATP resulted in detection of NH2Y radical (NH2Y) intermediates capable ofdNDP formation. In this study, the reaction of F3Y356-α2, α2, CDP, and ATP has been examined by stopped-flow (SF) absorption and rapid freeze quench electron paramagnetic resonance spectroscopy and has failed to reveal any radical intermediates. The reaction of F3Y356-α2, CDP, and ATP has also been examined with NH2Y731-α2 (or NH2Y735-α2) by SF kinetics from pH 6.5 to 9.2 and exhibited rate constants for NH2Y formation that support a change in the rate-limiting step at elevated pH. The results together with kinetic simulations provide a guide for future studies to detect radical intermediates in the pathway. [ABSTRACT FROM AUTHOR]

The class Ib ribonucleotide reductase of Escherichia coli can initiate reduction of nucleotides to deoxynucleotides with either a Mn|||2-tyrosyl radical (Y·) or a Fe|||2-Y· cofactor in the NrdF subunit. Whereas Fe|||2-Y· can self-assemble from Fe||2-NrdF and O2, activation of Mn||2-NrdF requires a reduced flavoprotein, Nrdl, proposed to form the oxidant for cofactor assembly by reduction of O2. The crystal structures reported here of E. coli Mn||2-NrdF and Fe||2-NrdF reveal different coordination environments, suggesting distinct initial binding sites for the oxidants during cofactor activation. In the structures of Mn||2-NrdF jn complex with reduced and oxidized Nrdl, a continuous channel connects the Nrdl flavin cofactor to the NrdF Mn||2 active site. Crystallographic detection of a putative peroxide in this channel supports the proposed mechanism of Mn|||2-Y· cofactor assembly. [ABSTRACT FROM AUTHOR]

E. coli ribonucleotide reductase catalyzes the reduction of nucleoside 5′-diphosphates into 2′-deoxynucleotides and is composed of two subunits: α2 and β2. During turnover, a stable tyrosyl radical (Y•) at Y122-β2 reversibly oxidizes C439 in the active site of α2. This radical propagation step is proposed to occur over 35 Å, to use specific redox-active tyrosines (Y122 and Y356 in β2, Y731 and Yβ in α2), and to involve proton-coupled electron transfer (PCET). 3-Nitrotyrosine (NO2Y, pKa 7.1) has been incorporated in place of Y122, Y731, and Y730 to probe how the protein environment perturbs each pKa in the presence of the second subunit, substrate (S), and allosteric effector (E). The activity of each mutant is <4 × 10-3 that of the wild-type (wt) subunit. The [NO2Y730]-α2 and [NO2Y731]-α2 each exhibit a pKa of 7.8-8.0 with E and E/β2. The pKa of [NO2Y730]-α2 is elevated to 8.2-8.3 in the S/E/β2 complex, whereas no further perturbation is observed for [NO2Y731]-α2. Mutations in pathway residues adjacent to the NOαY that disrupt H-bonding minimally perturb its pKa. The pKa of NO2Y122-β2 alone or with α2/S/E is >9.6. X-ray crystal structures have been obtained for all [NO2Y]-α2 mutants (2.1-3.1 Å resolution), which show minimal structural perturbation compared to wt-α2. Together with the pKa of the previously reported NO2Y356-β2 (7.5 in the α2/S/E complex; Yee, C. et al. Biochemistry 2003, 42, 14541-14552), these studies provide a picture of the protein environment of the ground state at each Y in the PCET pathway, and are the starting point for understanding differences in PCET mechanisms at each residue in the pathway. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductase (RNR, 76 kDa) from Lactobacillus leichmannii is a class 11 RNR that requires adenosylcobalamin (AdoCbl) as a cofactor. It catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is 100% itiactivated by I equiv of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F2C'I'P) in <2 mm. Sephadex 0-50 chromatography of the inactivation reaction mixture for 2 mm revealed that 0.47 equiv of a sugar moiety is covalently bound to RNR and 0.25 equiv of a cobalt(III) corrin is tightly associated, likely through a covalent interaction with C419 (Co-S) in the active site of RNR [Lohman, G. J. S., and Stubbe, J. (2010) Biochemistry 49, DOl: 10.1021 /bi902132u]. After I h, a similar experiment revealed 0.45 equiv Of the Co-S adduct associated with the protein. Thus, at least two pathways are associated with RNR inactivation: one associated with alkylation by the sugar of F2CTP and the second with AdoCbl destruction. To determine the fate of [I'-3H]F2CTP in the latter pathway, the reaction mixture at 2 mm was reduced with NaBH4 (NaB2H4) and the protein separated from the small molecules using a centrifugation device. The small molecules were dephosphorylatedand analyzed by HPLC to reveal 0.25 equiv of a stereoisomer of cytidine, characterized by mass spectrometry and NMR spectroscopy, indicating the trapped nucleotide had lost both of its fluorides and gained an o,ygen. High-field ENDOR studies with [1'H]F2CTP from the reaction quenched at 30 s revealed a radical that is nucleotide-based. The relationship between this radical and the trapped cytidmne analogue provides insight into the nonalkylative pathway for RNR inactivation relative to the alkylative pathway. [ABSTRACT FROM AUTHOR]

In the fourth step of the purine biosynthetic pathway, formyl glycinamide ribonucleotide (FGAR) amidotransferase, also known as PurL, catalyzes the conversion of FGAR, ATP, and glutamine to formyl glycinamidine ribonucleotide (FGAM), ADP, Pi, and glutamate. Two forms of PurL have been characterized, large and small. Large PurL, present in most Gram-negative bacteria and eukaryotes, consists of a single polypeptide chain and contains three major domains: the N-terminal domain, the FGAM synthetase domain, and the glutaminase domain, with a putative ammonia channel located between the active sites of the latter two. Small PuRL, present in Gram-positive bacteria and archaea, is structurally homologous to the FGAM synthetase domain of large PurL, and forms a complex with two additional gene products, PurQ and PurS. The structure of the PurS dimer is homologous with the N-terminal domain of large PuRL, while PurQ, whose structure has not been reported, contains the glutaminase activity. In Bacillus subtilis, the formation of the PurLQS complex is dependent on glutamine and ADP and has been demonstrated by size-exclusion chromatography. In this work, a structure of the PurLQS complex from Thermotoga maritima is described revealing a 2:1:1 stoichiometry of PurS:Q:L, respectively. The conformational changes observed in TmPurL upon complex formation elucidate the mechanism of metabolite-mediated recruitment of PurQ and PutS. The flexibility of the PurS dimer is proposed to play a role in the activation of the complex and the formation of the ammonia channel. A potential path for the ammonia channel is identified. [ABSTRACT FROM AUTHOR]

The article discusses a study which observes a transient tyrosine radical competent for initiating turnover in a photochemical ribonucleotide reductase. Proton-coupled electron transfer is vital to the generation and transport of amino acid radicals within class I Escherichia coli ribonucleotide reductase (RNR). RNR catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates. It also plays a vital role in deoxyribonucleic acid replication and repair.

Ribonucleotide reductases (RNRs) convert ribonucleotides into deoxyribonucleotides, a reaction essential for DNA replication and repair. Human RNR requires two subunits for activity, the α subunit contains the active site, and the β subunit houses the radical cofactor. Here, we present a 3.3-Å resolution structure by cryo-electron microscopy (EM) of a dATP-inhibited state of human RNR. This structure, which was determined in the presence of substrate CDP and allosteric regulators ATP and dATP, has three α2 units arranged in an α6 ring. At near-atomic resolution, these data provide insight into the molecular basis for CDP recognition by allosteric specificity effectors dATP/ATP. Additionally, we present lower-resolution EM structures of human α6 in the presence of both the anticancer drug clofarabine triphosphate and β2. Together, these structures support a model for RNR inhibition in which β2 is excluded from binding in a radical transfer competent position when α exists as a stable hexamer. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductases (RNRs) are responsible for all de novo biosynthesis of DNA precursors in nature by catalyzing the conversion of ribonucleotides to deoxyribonucleotides. Because of its essential role in cell division, human RNR is a target for a number of anticancer drugs in clinical use. Like other class Ia RNRs, human RNR requires both a radical-generation subunit (β) and nucleotide-binding subunit (α) for activity. Because of their complex dependence on allosteric effectors, however, the active and inactive quaternary forms of many class Ia RNRs have remained in question. Here, we present an X-ray crystal structure of the human α subunit in the presence of inhibiting levels of dATP, depicting a ring-shaped hexamer (α6) where the active sites line the inner hole. Surprisingly, our small-angle X-ray scattering (SAXS) results indicate that human α forms a similar hexamer in the presence of ATP, an activating effector. In both cases, α6 is assembled from dimers (α2) without a previously proposed tetramer intermediate (α4). However, we show with SAXS and electron microscopy that at millimolar ATP, the ATP-induced α6 can further interconvert with higher-order filaments. Differences in the dATP- and ATP-induced α6 were further examined by SAXS in the presence of the β subunit and by activity assays as a function of ATP or dATP. Together, these results suggest that dATP-induced α6 is more stable than the ATP-induced α6 and that stabilization of this ring-shaped configuration provides a mechanism to prevent access of the β subunit to the active site of α. [ABSTRACT FROM AUTHOR]

Summary: Clofarabine (ClF) is a drug used in the treatment of leukemia. One of its primary targets is human ribonucleotide reductase (hRNR), a dual-subunit, (α2)m(β2)n, regulatory enzyme indispensable in de novo dNTP synthesis. We report that, in live mammalian cells, ClF targets hRNR by converting its α-subunit into kinetically stable hexamers. We established mammalian expression platforms that enabled isolation of functional α and characterization of its altered oligomeric associations in response to ClF treatment. Size exclusion chromatography and electron microscopy documented persistence of in-cell-assembled-α6. Our data validate hRNR as an important target of ClF, provide evidence that in vivo α''s quaternary structure can be perturbed by a nonnatural ligand, and suggest small-molecule-promoted, persistent hexamerization as a strategy to modulate hRNR activity. These studies lay foundations for documentation of RNR oligomeric state within a cell. [Copyright &y& Elsevier]

Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides using a diferric tyrosyl radical (Y122•) cofactor in β2 to initiate catalysis in α2. Each turnover requires reversible long-range proton-coupled electron transfer (PCET) over 35 Å between the two subunits by a specific pathway (Y122• [W48?] Y356 within β to Y731 Y730 C439 within α). Previously, we reported that a β2 mutant with 3-nitrotyrosyl radical (NO2Y•; 1.2 radicals/β2) in place of Y122• in the presence of α2, CDP, and ATP catalyzes formation of 0.6 equiv of dCDP and accumulates 0.6 equiv of a new Y• proposed to be located on Y356 in β2. We now report three independent methods that establish that Y356 is the predominant location (85-90%) of the radical, with the remaining 10-15% delocalized onto Y731 and Y730 in α2. Pulsed electron-electron double-resonance spectroscopy on samples prepared by rapid freeze quench (RFQ) methods identified three distances: 30 ± 0.4 Å (88% ± 3%) and 33 ± 0.4 and 38 ± 0.5 Å (12% ± 3%) indicative of NO2Y122•-Y356•, NO2Y122•-NO2Y122•, and NO2Y122•-Y731(730)•, respectively. Radical distribution in α2 was supported by RFQ electron paramagnetic resonance (EPR) studies using Y731(3,5-F2Y) or Y730(3,5-F2Y)-α2, which revealed F2Y•, studies using globally incorporated [β-2H2]Y-α2, and analysis using parameters obtained from 140 GHz EPR spectroscopy. The amount of Y• delocalized in α2 from these two studies varied from 6% to 15%. The studies together give the first insight into the relative redox potentials of the three transient Y• radicals in the PCET pathway and their conformations. [ABSTRACT FROM AUTHOR]

E. coil ribonucleotide reductase (ANA) catalyzes the conversion of nucleotides to deoxynucleotides, a process that requires long-range radical transfer over 35 Å from a tyrosyl radical (Y122•) within the β2 subunit to a cysteine residue (C439) within the α2 subunit. The radical transfer step is proposed to occur by proton-coupled electron transfer via a specific pathway consisting of Y122 → W48 → Y356 in β2, across the subunit interface to Y731 → Y730 → C439 in α2. Using the suppressor tRNAlaminoacyl-tRNA synthetase (AS) methodology, 3-aminotyrosine has been incorporated into position 730 in α2. Incubation of this mutant with β2, substrate, and allosteric effector resulted in loss of the Y122• and formation of a new radical, previously proposed to be a 3-aminotyrosyl radical (NH2Y•). In the current study [15N]- and [14N]-NH2Y730• have been generated in H2O and D2O and characterized by continuous wave 9 3Hz EPA and pulsed EPA spectroscopies at 9, 94, and 180 3Hz. The data give insight into the electronic and molecular structure of NH2Y730•. The g tensor (gx = 2.0052, gy = 2.0042, gz = 2.0022), the orientation of the β-protons, the hybridization of the amine nitrogen, and the orientation of the amino protons relative to the plane of the aromatic ring were determined. The hyperfine coupling constants and geometry of the NH2 moiety are consistent with an intramolecular hydrogen bond within NH2Y730•. This analysis is an essential first step in using the detailed structure of NH2Y730• to formulate a model for a PCET mechanism within α2 and for use of NH2Y in other systems where transient Y•s participate in catalysis. [ABSTRACT FROM AUTHOR]

Photochemical ribonucleotide reductases (photoRNRs) have been developed to study the proton-coupled electron transfer (PCET) mechanism of radical transport in Escherichia coli class I ribonucleotide reductase (RNR). The transport of the effective radical occurs along several conserved aromatic residues across two subunits: β2(.Y122 → W48 → Y356) → α2(Y731 → Y730 → C439). The current model for RNR activity suggests that radical transport is strongly controlled by conformational gating. The C-terminal tail peptide (Y-βC19) of β2 is the binding determinant of β2 to β2 and contains the redox active Y356 residue. A photoRNR has been generated synthetically by appending a Re(bpy)(CO)3CN ([Re]) photo-oxidant next to Y356 of the 20-mer peptide. Emission from the [Re] center dramatically increases upon peptide binding, serving as a probe for conformational dynamics and the protonation state of Y356. The diffusion coefficient of [Re]-Y-βC19 has been measured (kd1 = 6.1 x 10-7 cm-1 s-1), along with the dissociation rate constant for the [Re]-Y-βC19-α2 complex (7000 s-1 > koff > 400 s-1). Results from detailed time-resolved emission and absorption spectroscopy reveal biexponential kinetics, suggesting a large degree of conformational flexibility in the [Re]-Y-βC19-α2 complex that engenders partitioning of the N-terminus of the peptide into both bound and solvent-exposed fractions. [ABSTRACT FROM AUTHOR]

The Eschericliia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleotides and requires a diferric-tyrosyl radical cofactor for catalysis. RNR is composed of a 1:1 complex of two homodimeric subunits: a and β. Incubation of the E441Q-α mutant RNR with substrate CDP and allosteric effector TTP results in loss of the tyrosyl radical and formation of two new radicals on the 200 ms to min time scale. The first radical was previously established by stopped flow UV/vis spectroscopy and pulsed high field EPR spectroscopy to be a disulfide radical anion. The second radical was proposed to be a 4'-radical of a 3'-keto-2'-deoxycytidine 5'-diphosphate. To identify the structure of the nucleotide radical [1'-2H], [2'-2H], [4'-2H], [5'-2H], [U-13C, 15N], [U-15N], and [5,6 -2H] CDP and [β-2H] cysteine-α were synthesized and incubated with E441Q-α2β2 and TTP. The nucleotide radical was examined by 9 GHz and 140 GHz pulsed EPR spectroscopy and 35GHz ENDOR spectroscopy. Substitution of 2H at C4' and C1' altered the observed hyperfine interactions of the nucleotide radical and established that the observed structure was not that predicted. DFT calculations (B3LYP/IGLO-lll/B3LYP/ TZVP) were carried out in an effort to recapitulate the spectroscopic observations and lead to a new structure consistent with all of the experimental data. The results indicate, unexpectedly, that the radical is a semidione nucleotide radical of cytidine 5'-diphosphate. The relationship of this radical to the disulfide radical anion is discussed. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. The class I RNRs are composed of a 1:1 complex of two homodimeric subunits: a and β. β contains the diferric-tyrosyl radical (Y') cofactor essential for the reduction process. In vivo, the mechanism of Y• regeneration from the diferric-β2 (met-β2) or apo-β2 is still unclear. Y• regenerations from met-β2 and apo-β2 have been designated the maintenance and biosynthetic pathways, respectively. To understand these two pathways, 181 genomes that contain nrdAnrdB (genes encoding α and β) were examined. In 29% of the cases, an open reading frame annotated 2Fe2S ferredoxin (YfaE in Escherichia coli) is located next to nrdB. Thus, YfaE has been cloned, expressed, resolubilized, reconstituted anaerobically with Fe2+, Fe3+, and S2-, and characterized by. Mössbauer, EPR, and visible spectroscopies. Titration of met-β2 with [2Fe2S]1+-YfaE anaerobically results in the formation of an equilibrium mixture of diferrous-β2 and [2Fe2S]2+-YfaE with one Fe reduced/YfaE oxidized. At the end point of the titration, O2 is added to the mixture and the diferrous-β2 rapidly undergoes reaction to form the diferric-Y• with a stoichiometry of 2Fe/Y• and a specific activity correlated to the amount of Y•. The reducing equivalent required for diferric-Y• cofactor biosynthesis is supplied by/3. Under anaerobic conditions, stopped flow kinetics have been used to monitor the disappearance of the diferric cluster and the formation of [2Fe2S]2+-YfaE. The titrations and kinetic studies provide the first evidence for a protein involved in the maintenance pathway and likely the biosynthetic pathway. [ABSTRACT FROM AUTHOR]

The class I ribonucleotide reductases catalyze the conversion of nucleotides to deoxynucleotides and are composed of two subunits: R1 and R2. R1 contains the site for nucleotide reduction and the sites that control substrate specificity and the rate of reduction. R2 houses the essential diferric-tyrosyl radical (Y•) cofactor. In Saccharomyces cerevisiae, two R1s, αn and α'n, have been identified, while R2 is a heterodimer (ββ). β' cannot bind iron and generate the Y• consequently, the maximum amount of Y• per ββ' is 1. To determine the cofactor stoichiometry in vivo, a FLAG-tagged β (FLAGβ) was constructed and integrated into the genome of Y300 (MHY343). This strain facilitated the rapid isolation of endogenous levels of FLAGββ' by immunoaffinity chromatography, which was found to have 0.45 ± 0.08 Y•/FLAGββ' and a specific activity of 2.3 ± 0.5 μmol min-1 mg-1 FLAGββ' isolated from MMS-treated MHY343 cells or cells containing a deletion of the transcriptional repressor gene CRT1 also gave a Y•/FLAGββ' ratio of 0.5. To determine the Y•/ββ' ratio without R2 isolation, whole cell EPR and quantitative Western blots of β were performed using different strains and growth conditions. The wild-type (wt) strains gave a Y•/ββ' ratio of 0.83-0.89. The same strains either treated with MMS or containing a crt1Δ gave ratios between 0.49 and 0.72. Nucleotide reduction assays and quantitative Western blots from the same strains provided an independent measure and confirmation of the Y•/ββ' ratios. Thus, under normal growth conditions, the cell assembles stoichiometric amounts of Y• and modulation of Y• concentration is not involved in the regulation of RNR activity. [ABSTRACT FROM AUTHOR]

The class I ribonucleotide reductases (RNRs) are composed of two homodimeric subunits: R1 and R2. R2 houses a diferric-tyrosyl radical (Y·) cofactor. Saccharomyces cerevisiae has two R2s: Y2 (β2) and Y4 (β2'). Y4 is an unusual R2 because three residues required for iron binding have been mutated. While the heterodimer (ββ') is thought to be the active form, several rnr4Δ strains are viable. To resolve this paradox, N-terminally epitope-tagged β and β' were expressed in E. coli or integrated into the yeast genome. In vitro exchange studies reveal that when apo-(His6)-β2 (Hisβ2) is mixed with β'2, apo-Hisββ' forms quantitatively within 2 mm. In contrast, holo-ββ' fails to exchange with apo-Hisβ2 to form holo-Hisββ and β'2. Isolation of genomically encoded tagged β or β' from yeast extracts gave a 1:1 complex of β and β', suggesting that ββ' is the active form. The catalytic activity, protein concentrations, and Y content of the rnrΔΔ and wild type (wt) strains were compared to clarify the role of β' in vivo. The Y· content of rnr4Δ is 15-fold less than that of wt, consistent with the observed low activity of rnr4Δ extracts (<0.01 nmol min-1 mg-1) versus wt (0.06 ± 0.01 nmol min-1 mg-1). FLAGβ2 isolated from the rnr4zS strain has a specific activity of 2 nmol min-1 mg-1, similar to that of reconstituted apo- Hisβ2 (10 nmol min-1 mg-1), but significantly less than holo-Hisββ' (∼2000 nmol min-1 mg-1). These studies together demonstrate that β' plays a crucial role in cluster assembly in vitro and in vivo and that the active form of the yeast R2 is ββ'. [ABSTRACT FROM AUTHOR]

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides providing the monomeric precursors required for DNA replication and repair. The class I RNRs are composed of two homodimeric subunits: R1 and R2. R1 has the active site where nucleotide reduction occurs, and R2 contains the diiron tyrosyl radical (Y•) cofactor essential for radical initiation on R1. Mechanism-based inhibitors, such as 2'-azido-2'-deoxyuridine-5'-diphosphate (N³UDP), have provided much insight into the reduction mechanism. N³UDP is a stoichiometric inactivator that, upon interaction with RNR, results in loss of the Y• in R2 and formation of a nitrogen-centered radical (N•) covalently attached to C225 (R-S-N•-X) in the active site of R1. N2; is lost prior to N• formation, and after its formation, stoichiometric amounts of 2-methylene-3-furanone, pyrophosphate, and uracil are also generated. On the basis of the hyperfine interactions associated with N•, it was proposed that N• is also covalently attached to the nucleotide through either the oxygen of the 3'-OH (R-S-N•-O-R') or the 3'-C (R-S-N•-C-OH). To distinguish between the proposed structures, the inactivation was carried out with 3'-[17O]-N3UDP and N• was examined by 9 and 140 GHz EPR spectroscopy. Broadening of the N• signal was detected and the spectrum simulated to obtain the [17O] hypertine tensor. DFT calculations were employed to determine which structures are in best agreement with the simulated hypertine tensor and our previous ESEEM data. The results are most consistent with the R-S-N•-C-OH structure and provide evidence for the trapping of a 3'-ketonucleotide in the reduction process. [ABSTRACT FROM AUTHOR]

Class I ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides. Eukaryotic RNRs comprise two subunits, the R1 subunit, which contains substrate and allosteric effector binding sites, and the R2 subunit, which houses a catalytically essential diirontyrosyl radical cofactor. In Saccharomyces cerevisiae, there are two variants of the R2 subunit, called Rnr2 and Rnr4. Rnr4 is unique in that it lacks three iron-binding residues conserved in all other R2s. Nevertheless, Rnr4 is required to activate Rnr2, and the functional species in vivo is believed to be a heterodimeric complex between the two proteins. The crystal structures of the Rnr2 and Rnr4 homodimers have been determined and are compared to that of the heterodimer. The homodimers are very similar to the heterodimer and to mouse R2 in overall fold, but there are several key differences. In the Rnr2 homodimer, one of the iron-binding helices, helix αB, is not well-ordered. In the heterodimer, interactions with a loop region connecting Rnr4 helices αA and α3 stabilize this Rnr2 helix, which donates iron ligand Asp 145. Sequence differences between Rnr2 and Rnr4 prevent the same interactions from occurring in the Rnr2 homodimer. These findings provide a structural rationale for why the heterodimer is the preferred complex in vivo. The active-site region in the Rnr4 homodimer reveals interactions not apparent in the heterodimer, supporting previous conclusions that this subunit does not bind iron. When taken together, these results support a model in which Rnr4 stabilizes Rnr2 for cofactor assembly and activity. [ABSTRACT FROM AUTHOR]

The article examines the distance between residues involved in the radical propagation pathway of Escherichia (E.) coli ribonucleotide reductase (RNR). The researchers consider the docking model which is generated from the individual structures of α2 and ß2 based on shape and charge complementarities in analyzing the distances. Based on the results, the study shows the position of the critical Tyr pathway residues in the α2ß2 complex.

Focuses 2,3-difluorotyrosine at position 356 of ribonucleotide reductase R2. Probe of long-range proton-coupled electron transfer (PCET); Proposed PCET pathway and distanced based on crystal structures of R1 and R2.