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Ribonucleotide reductases (RNRs) are essential enzymes that catalyze the de novo transformation of nucleoside 5'-di(tri)phosphates [ND(T)Ps, where N is A, U, C, or G] to their corresponding deoxynucleotides. Despite the diversity of factors required for function and the low sequence conservation across RNRs, a unifying apparatus consolidating RNR activity is explored. We combine aspects of the protein subunit simplicity of class II RNR with a modified version of Escherichia coli class la photoRNRs that initiate radical chemistry with light to engineer a mimic of a class II enzyme. The design of this RNR involves fusing a truncated form of the active site containing α subunit with the functionally important C-terminal tail of the radical-generating β subunit to render a chimeric RNR. Inspired by a recent cryo-EM structure, a [Re] photooxidant is located adjacent to Y 356 [β], which is an essential component of the radical transport pathway in class I RNRs. Combination of this RNR photochimera with cytidine diphosphate (CDP), adenosine triphosphate (ATP), and light resulted in the generation of Y 356 • along with production of deoxycytidine diphosphate (dCDP) and cytosine. The photoproducts reflect an active site chemistry consistent with both the consensus mechanism of RNR and chemistry observed when RNR is inactivated by mechanism-based inhibitors in the active site. The enzymatic activity of the RNR photochimera in the absence of any β metallocofactor highlights the adaptability of the 10-stranded αβ barrel finger loop to support deoxynucleotide formation and accommodate the design of engineered RNRs., Competing Interests: Competing interests statement:The authors declare no competing interest.

Ribonucleotide reductases (RNRs) catalyze the conversion of all four ribonucleotides to deoxyribonucleotides and are essential for DNA synthesis in all organisms. The active form of E. coli Ia RNR is composed of two homodimers that form the active α 2 β 2 complex. Catalysis is initiated by long-range radical translocation over a ∼32 Å proton-coupled electron transfer (PCET) pathway involving Y356β and Y731α at the interface. Resolving the PCET pathway at the α/β interface has been a long-standing challenge due to the lack of structural data. Herein, molecular dynamics simulations based on a recently solved cryogenic-electron microscopy structure of an active α 2 β 2 complex are performed to examine the structure and fluctuations of interfacial water, as well as the hydrogen-bonding interactions and conformational motions of interfacial residues along the PCET pathway. Our free energy simulations reveal that Y731 is able to sample both a flipped-out conformation, where it points toward the interface to facilitate interfacial PCET with Y356, and a stacked conformation with Y730 to enable collinear PCET with this residue. Y356 and Y731 exhibit hydrogen-bonding interactions with interfacial water molecules and, in some conformations, share a bridging water molecule, suggesting that the primary proton acceptor for PCET from Y356 and from Y731 is interfacial water. The conformational flexibility of Y731 and the hydrogen-bonding interactions of both Y731 and Y356 with interfacial water and hydrogen-bonded water chains appear critical for effective radical translocation along the PCET pathway. These simulations are consistent with biochemical and spectroscopic data and provide previously unattainable atomic-level insights into the fundamental mechanism of RNR.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides (NDP) to deoxynucleotides (dNDP), in part, by controlling the ratios and quantities of dNTPs available for DNA replication and repair. The active form of Escherichia coli class Ia RNR is an asymmetric α 2 β 2 complex in which α 2 contains the active site and β 2 contains the stable diferric-tyrosyl radical cofactor responsible for initiating the reduction chemistry. Each dNDP is accompanied by disulfide bond formation. We now report that, under in vitro conditions, β 2 can initiate turnover in α 2 catalytically under both "one" turnover (no external reductant, though producing two dCDPs) and multiple turnover (with an external reductant) assay conditions. In the absence of reductant, rapid chemical quench analysis of a reaction of α 2 , substrate, and effector with variable amounts of β 2 (1-, 10-, and 100-fold less than α 2 ) yields 3 dCDP/α 2 at all ratios of α 2 :β 2 with a rate constant of 8-9 s -1 , associated with a rate-limiting conformational change. Stopped-flow fluorescence spectroscopy with a fluorophore-labeled β reveals that the rate constants for subunit association (163 ± 7 μM -1 s -1 ) and dissociation (75 ± 10 s -1 ) are fast relative to turnover, consistent with catalytic β 2 . When assaying in the presence of an external reducing system, the turnover number is dictated by the ratio of α 2 :β 2 , their concentrations, and the concentration and nature of the reducing system; the rate-limiting step can change from the conformational gating to a step or steps involving disulfide rereduction, dissociation of the inhibited α 4 β 4 state, or both. The issues encountered with E. coli RNR are likely of importance in all class I RNRs and are central to understanding the development of screening assays for inhibitors of these enzymes.

3-Aminotyrosine (NH 2 Y) has been a useful probe to study the role of redox active tyrosines in enzymes. This report describes properties of NH 2 Y of key importance for its application in mechanistic studies. By combining the tRNA/NH 2 Y-RS suppression technology with a model protein tailored for amino acid redox studies (α 3 X, X = NH 2 Y), the formal reduction potential of NH 2 Y 32 (O • /OH) ( E°' = 395 ± 7 mV at pH 7.08 ± 0.05) could be determined using protein film voltammetry. We find that the Δ E°' between NH 2 Y 32 (O • /OH) and Y 32 (O • /OH) when measured under reversible conditions is ∼300-400 mV larger than earlier estimates based on irreversible voltammograms obtained on aqueous NH 2 Y and Y. We have also generated D 6 -NH 2 Y 731 -α2 of ribonucleotide reductase (RNR), which when incubated with β2/CDP/ATP generates the D 6 -NH 2 Y 731 • -α2/β2 complex. By multifrequency electron paramagnetic resonance (35, 94, and 263 GHz) and 34 GHz 1 H ENDOR spectroscopies, we determined the hyperfine coupling (hfc) constants of the amino protons that establish RNH 2 • planarity and thus minimal perturbation of the reduction potential by the protein environment. The amount of Y in the isolated NH 2 Y-RNR incorporated by infidelity of the tRNA/NH 2 Y-RS pair was determined by a generally useful LC-MS method. This information is essential to the utility of this NH 2 Y probe to study any protein of interest and is employed to address our previously reported activity associated with NH 2 Y-substituted RNRs.

The reaction catalyzed by E. coli ribonucleotide reductase (RNR) composed of α and β subunits that form an active α2β2 complex is a paradigm for proton-coupled electron transfer (PCET) processes in biological transformations. β2 contains the diferric tyrosyl radical (Y 122 ·) cofactor that initiates radical transfer (RT) over 35 Å via a specific pathway of amino acids (Y 122 · ⇆ [W 48 ] ⇆ Y 356 in β2 to Y 731 ⇆ Y 730 ⇆ C 439 in α2). Experimental evidence exists for colinear and orthogonal PCET in α2 and β2, respectively. No mechanistic model yet exists for the PCET across the subunit (α/β) interface. Here, we report unique EPR spectroscopic features of Y 356 ·-β, the pathway intermediate generated by the reaction of 2,3,5-F 3 Y 122 ·-β2/CDP/ATP with wt-α2, Y 731 F-α2, or Y 730 F-α2. High field EPR (94 and 263 GHz) reveals a dramatically perturbed g tensor. [ 1 H] and [ 2 H]-ENDOR reveal two exchangeable H bonds to Y 356 ·: a moderate one almost in-plane with the π-system and a weak one. DFT calculation on small models of Y· indicates that two in-plane, moderate H bonds (r O-H ∼1.8-1.9 Å) are required to reproduce the g x value of Y 356 · (wt-α2). The results are consistent with a model, in which a cluster of two, almost symmetrically oriented, water molecules provide the two moderate H bonds to Y 356 · that likely form a hydrogen bond network of water molecules involved in either the reversible PCET across the subunit interface or in H + release to the solvent during Y 356 oxidation.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleoside diphosphate substrates (S) to deoxynucleotides with allosteric effectors (e) controlling their relative ratios and amounts, crucial for fidelity of DNA replication and repair. Escherichia coli class Ia RNR is composed of α and β subunits that form a transient, active α2β2 complex. The E. coli RNR is rate-limited by S/e-dependent conformational change(s) that trigger the radical initiation step through a pathway of 35 Å across the subunit (α/β) interface. The weak subunit affinity and complex nucleotide-dependent quaternary structures have precluded a molecular understanding of the kinetic gating mechanism(s) of the RNR machinery. Using a docking model of α2β2 created from X-ray structures of α and β and conserved residues from a new subclassification of the E. coli Ia RNR (Iag), we identified and investigated four residues at the α/β interface (Glu 350 and Glu 52 in β2 and Arg 329 and Arg 639 in α2) of potential interest in kinetic gating. Mutation of each residue resulted in loss of activity and with the exception of E52Q-β2, weakened subunit affinity. An RNR mutant with 2,3,5-trifluorotyrosine radical (F 3 Y 122 • ) replacing the stable Tyr 122 • in WT-β2, a mutation that partly overcomes conformational gating, was placed in the E52Q background. Incubation of this double mutant with His 6 -α2/S/e resulted in an RNR capable of catalyzing pathway-radical formation (Tyr 356• -β2), 0.5 eq of dCDP/F 3 Y 122 • , and formation of an α2β2 complex that is isolable in pulldown assays over 2 h. Negative stain EM images with S/e (GDP/TTP) revealed the uniformity of the α2β2 complex formed., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Escherichia coli class Ia ribonucleotide reductase (RNR) converts ribonucleotides to deoxynucleotides. A diferric-tyrosyl radical (Y 122 •) in one subunit (β2) generates a transient thiyl radical in another subunit (α2) via long-range radical transport (RT) through aromatic amino acid residues (Y 122 ⇆ [W 48 ] ⇆ Y 356 in β2 to Y 731 ⇆ Y 730 ⇆ C 439 in α2). Equilibration of Y 356 •, Y 731 •, and Y 730 • was recently observed using site specifically incorporated unnatural tyrosine analogs; however, equilibration between Y 122 • and Y 356 • has not been detected. Our recent report of Y 356 • formation in a kinetically and chemically competent fashion in the reaction of β2 containing 2,3,5-trifluorotyrosine at Y 122 (F 3 Y 122 •-β2) with α2, CDP (substrate), and ATP (effector) has now afforded the opportunity to investigate equilibration of F 3 Y 122 • and Y 356 •. Incubation of F 3 Y 122 •-β2, Y 731 F-α2 (or Y 730 F-α2), CDP, and ATP at different temperatures (2-37 °C) provides ΔE°'(F 3 Y 122 •-Y 356 •) of 20 ± 10 mV at 25 °C. The pH dependence of the F 3 Y 122 • ⇆ Y 356 • interconversion (pH 6.8-8.0) reveals that the proton from Y 356 is in rapid exchange with solvent, in contrast to the proton from Y 122 . Insertion of 3,5-difluorotyrosine (F 2 Y) at Y 356 and rapid freeze-quench EPR analysis of its reaction with Y 731 F-α2, CDP, and ATP at pH 8.2 and 25 °C shows F 2 Y 356 • generation by the native Y 122 •. F n Y-RNRs (n = 2 and 3) together provide a model for the thermodynamic landscape of the RT pathway in which the reaction between Y 122 and C 439 is ∼200 meV uphill.

Fluorinated tyrosines (FnY's, n = 2 and 3) have been site-specifically incorporated into E. coli class Ia ribonucleotide reductase (RNR) using the recently evolved M. jannaschii Y-tRNA synthetase/tRNA pair. Class Ia RNRs require four redox active Y's, a stable Y radical (Y·) in the β subunit (position 122 in E. coli), and three transiently oxidized Y's (356 in β and 731 and 730 in α) to initiate the radical-dependent nucleotide reduction process. FnY (3,5; 2,3; 2,3,5; and 2,3,6) incorporation in place of Y122-β and the X-ray structures of each resulting β with a diferric cluster are reported and compared with wt-β2 crystallized under the same conditions. The essential diferric-FnY· cofactor is self-assembled from apo FnY-β2, Fe(2+), and O2 to produce ∼1 Y·/β2 and ∼3 Fe(3+)/β2. The FnY· are stable and active in nucleotide reduction with activities that vary from 5% to 85% that of wt-β2. Each FnY·-β2 has been characterized by 9 and 130 GHz electron paramagnetic resonance and high-field electron nuclear double resonance spectroscopies. The hyperfine interactions associated with the (19)F nucleus provide unique signatures of each FnY· that are readily distinguishable from unlabeled Y·'s. The variability of the abiotic FnY pKa's (6.4 to 7.8) and reduction potentials (-30 to +130 mV relative to Y at pH 7.5) provide probes of enzymatic reactions proposed to involve Y·'s in catalysis and to investigate the importance and identity of hopping Y·'s within redox active proteins proposed to protect them from uncoupled radical chemistry.

The Escherichia coli class Ia ribonucleotide reductase (RNR) achieves forward and reverse proton-coupled electron transfer (PCET) over a pathway of redox active amino acids (β-Y122 ⇌ β-Y356 ⇌ α-Y731 ⇌ α-Y730 ⇌ α-C439) spanning ∼35 Å and two subunits every time it turns over. We have developed photoRNRs that allow radical transport to be phototriggered at tyrosine (Y) or fluorotyrosine (FnY) residues along the PCET pathway. We now report a new photoRNR in which photooxidation of a tryptophan (W) residue replacing Y356 within the α/β subunit interface proceeds by a stepwise ET/PT (electron transfer then proton transfer) mechanism and provides an orthogonal spectroscopic handle with respect to radical pathway residues Y731 and Y730 in α. This construct displays an ∼3-fold enhancement in photochemical yield of W(•) relative to F3Y(•) and a ∼7-fold enhancement relative to Y(•). Photogeneration of the W(•) radical occurs with a rate constant of (4.4 ± 0.2) × 10(5) s(-1), which obeys a Marcus correlation for radical generation at the RNR subunit interface. Despite the fact that the Y → W variant displays no enzymatic activity in the absence of light, photogeneration of W(•) within the subunit interface results in 20% activity for turnover relative to wild-type RNR under the same conditions.

Activation of the diferrous center of the β2 (R2) subunit of the class 1a Escherichia coli ribonucleotide reductases by reaction with O2 followed by one-electron reduction yields a spin-coupled, paramagnetic Fe(III)/Fe(IV) intermediate, denoted X, whose identity has been sought by multiple investigators for over a quarter of a century. To determine the composition and structure of X, the present study has applied (57)Fe, (14,15)N, (17)O, and (1)H electron nuclear double resonance (ENDOR) measurements combined with quantitative measurements of (17)O and (1)H electron paramagnetic resonance line-broadening studies to wild-type X, which is very short-lived, and to X prepared with the Y122F mutant, which has a lifetime of many seconds. Previous studies have established that over several seconds the as-formed X(Y122F) relaxes to an equilibrium structure. The present study focuses on the relaxed structure. It establishes that the inorganic core of relaxed X has the composition [(OH(-))Fe(III)-O-Fe(IV)]: there is no second inorganic oxygenic bridge, neither oxo nor hydroxo. Geometric analysis of the (14)N ENDOR data, together with recent extended X-ray absorption fine structure measurements of the Fe-Fe distance (Dassama, L. M.; et al. J. Am. Chem. Soc. 2013, 135, 16758), supports the view that X contains a "diamond-core" Fe(III)/Fe(IV) center, with the irons bridged by two ligands. One bridging ligand is the oxo bridge (OBr) derived from O2 gas. Given the absence of a second inorganic oxygenic bridge, the second bridging ligand must be protein derived, and is most plausibly assigned as a carboxyl oxygen from E238.

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides in all organisms. In all Class Ia RNRs, initiation of nucleotide diphosphate (NDP) reduction requires a reversible oxidation over 35 Å by a tyrosyl radical (Y122•, Escherichia coli) in subunit β of a cysteine (C439) in the active site of subunit α. This radical transfer (RT) occurs by a specific pathway involving redox active tyrosines (Y122 ⇆ Y356 in β to Y731 ⇆ Y730 ⇆ C439 in α); each oxidation necessitates loss of a proton coupled to loss of an electron (PCET). To study these steps, 3-aminotyrosine was site-specifically incorporated in place of Y356-β, Y731- and Y730-α, and each protein was incubated with the appropriate second subunit β(α), CDP and effector ATP to trap an amino tyrosyl radical (NH2Y•) in the active α2β2 complex. High-frequency (263 GHz) pulse electron paramagnetic resonance (EPR) of the NH2Y•s reported the gx values with unprecedented resolution and revealed strong electrostatic effects caused by the protein environment. (2)H electron-nuclear double resonance (ENDOR) spectroscopy accompanied by quantum chemical calculations provided spectroscopic evidence for hydrogen bond interactions at the radical sites, i.e., two exchangeable H bonds to NH2Y730•, one to NH2Y731• and none to NH2Y356•. Similar experiments with double mutants α-NH2Y730/C439A and α-NH2Y731/Y730F allowed assignment of the H bonding partner(s) to a pathway residue(s) providing direct evidence for colinear PCET within α. The implications of these observations for the PCET process within α and at the interface are discussed.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. Active E. coli class Ia RNR is an α2β2 complex that undergoes reversible, long-range proton-coupled electron transfer (PCET) over a pathway of redox active amino acids (β-Y122 → [β-W48] → β-Y356 → α-Y731 → α-Y730 → α-C439) that spans ∼35 Å. To unmask PCET kinetics from rate-limiting conformational changes, we prepared a photochemical RNR containing a [Re(I)] photooxidant site-specifically incorporated at position 355 ([Re]-β2), adjacent to PCET pathway residue Y356 in β. [Re]-β2 was further modified by replacing Y356 with 2,3,5-trifluorotyrosine to enable photochemical generation and spectroscopic observation of chemically competent tyrosyl radical(s). Using transient absorption spectroscopy, we compare the kinetics of Y· decay in the presence of substrate and wt-α2, Y731F-α2 ,or C439S-α2, as well as with 3'-[(2)H]-substrate and wt-α2. We find that only in the presence of wt-α2 and the unlabeled substrate do we observe an enhanced rate of radical decay indicative of forward radical propagation. This observation reveals that cleavage of the 3'-C-H bond of substrate by the transiently formed C439· thiyl radical is rate-limiting in forward PCET through α and has allowed calculation of a lower bound for the rate constant associated with this step of (1.4 ± 0.4) × 10(4) s(-1). Prompting radical propagation with light has enabled observation of PCET events heretofore inaccessible, revealing active site chemistry at the heart of RNR catalysis.

The class III ribonucleotide reductases (RNRs) are glycyl radical (G•) enzymes that provide the balanced pool of deoxynucleotides required for DNA synthesis and repair in many facultative and obligate anaerobic bacteria and archaea. Unlike the class I and II RNRs, where reducing equivalents for the reaction are delivered by a redoxin (thioredoxin, glutaredoxin, or NrdH) via a pair of conserved active site cysteines, the class III RNRs examined to date use formate as the reductant. Here, we report that reaction of the Escherichia coli class III RNR with CTP (substrate) and ATP (allosteric effector) in the absence of formate leads to loss of the G• concomitant with stoichiometric formation of a new radical species and a "trapped" cytidine derivative that can break down to cytosine. Addition of formate to the new species results in recovery of 80% of the G• and reduction of the cytidine derivative, proposed to be 3'-keto-deoxycytidine, to dCTP and a small amount of cytosine. The structure of the new radical has been identified by 9.5 and 140 GHz EPR spectroscopy on isotopically labeled varieties of the protein to be a thiosulfuranyl radical [RSSR2]•, composed of a cysteine thiyl radical stabilized by an interaction with a methionine residue. The presence of a stable radical species on the reaction pathway rationalizes the previously reported [(3)H]-(k(cat)/K(M)) isotope effect of 2.3 with [(3)H]-formate, requiring formate to exchange between the active site and solution during nucleotide reduction. Analogies with the disulfide anion radical proposed to provide the reducing equivalent to the 3'-keto-deoxycytidine intermediate by the class I and II RNRs provide further evidence for the involvement of thiyl radicals in the reductive half-reaction catalyzed by all RNRs.

The reversible Y-O•/Y-OH redox properties of the α3Y model protein allow access to the electrochemical and thermodynamic properties of 3,5-difluorotyrosine. The unnatural amino acid has been incorporated at position 32, the dedicated radical site in α3Y, by in vivo nonsense codon suppression. Incorporation of 3,5-difluorotyrosine gives rise to very minor structural changes in the protein scaffold at pH values below the apparent pK (8.0±0.1) of the unnatural residue. Square-wave voltammetry on α3(3,5)F2Y provides an E°'(Y-O•/Y-OH) of 1026±4 mV versus the normal hydrogen electrode (pH 5.70±0.02) and shows that the fluoro substitutions lower the E°' by -30±3 mV. These results illustrate the utility of combining the optimized α3Y tyrosine radical system with in vivo nonsense codon suppression to obtain the formal reduction potential of an unnatural aromatic residue residing within a well-structured protein. It is further observed that the protein E°' values differ significantly from peak potentials derived from irreversible voltammograms of the corresponding aqueous species. This is notable because solution potentials have been the main thermodynamic data available for amino acid radicals. The findings in this paper are discussed relative to recent mechanistic studies of the multistep radical-transfer process in Escherichia coli ribonucleotide reductase site-specifically labeled with unnatural tyrosine residues.

Ribonucleotide reductases (RNRs) catalyze the conversionof nucleotides to 2'-deoxynucleotides and are classified on the basis of the metallo-cofactor used to conduct this chemistry. The class Ia RNRs initiate nucleotide reduction when a stable diferric-tyrosyl radical (Y•, t1/2 of 4 days at 4 °C) cofactor in the β2 subunit transiently oxidizes a cysteine to a thiyl radical (S•) in the active site of the α2 subunit. In the active α2β2 complex of the class Ia RNR from E. coli , researchers have proposed that radical hopping occurs reversibly over 35 Å along a specific pathway comprised of redox-active aromatic amino acids: Y122• ↔ [W48?] ↔ Y356 in β2 to Y731 ↔ Y730 ↔ C439 in α2. Each step necessitates a proton-coupled electron transfer (PCET). Protein conformational changes constitute the rate-limiting step in the overall catalytic scheme and kinetically mask the detailed chemistry of the PCET steps. Technology has evolved to allow the site-selective replacement of the four pathway tyrosines with unnatural tyrosine analogues. Rapid kinetic techniques combined with multifrequency electron paramagnetic resonance, pulsed electron-electron double resonance, and electron nuclear double resonance spectroscopies have facilitated the analysis of stable and transient radical intermediates in these mutants. These studies are beginning to reveal the mechanistic underpinnings of the radical transfer (RT) process. This Account summarizes recent mechanistic studies on mutant E. coli RNRs containing the following tyrosine analogues: 3,4-dihydroxyphenylalanine (DOPA) or 3-aminotyrosine (NH2Y), both thermodynamic radical traps; 3-nitrotyrosine (NO2Y), a thermodynamic barrier and probe of local environmental perturbations to the phenolic pKa; and fluorotyrosines (FnYs, n = 2 or 3), dual reporters on local pKas and reduction potentials. These studies have established the existence of a specific pathway spanning 35 Å within a globular α2β2 complex that involves one stable (position 122) and three transient (positions 356, 730, and 731) Y•s. Our results also support that RT occurs by an orthogonal PCET mechanism within β2, with Y122• reduction accompanied by proton transfer from an Fe1-bound water in the diferric cluster and Y356 oxidation coupled to an off-pathway proton transfer likely involving E350. In α2, RT likely occurs by a co-linear PCET mechanism, based on studies of light-initiated radical propagation from photopeptides that mimic the β2 subunit to the intact α2 subunit and on [(2)H]-ENDOR spectroscopic analysis of the hydrogen-bonding environment surrounding a stabilized NH2Y• formed at position 730. Additionally, studies on the thermodynamics of the RT pathway reveal that the relative reduction potentials decrease according to Y122 < Y356 < Y731 ≈ Y730 ≤ C439, and that the pathway in the forward direction is thermodynamically unfavorable. C439 oxidation is likely driven by rapid, irreversible loss of water during the nucleotide reduction process. Kinetic studies of radical intermediates reveal that RT is gated by conformational changes that occur on the order of >100 s(-1) in addition to the changes that are rate-limiting in the wild-type enzyme (∼10 s(-1)). The rate constant of one of the PCET steps is ∼10(5) s(-1), as measured in photoinitiated experiments.

Substrate turnover in class Ia ribonucleotide reductase (RNR) requires reversible radical transport across two subunits over 35 Å, which occurs by a multistep proton-coupled electron-transfer mechanism. Using a photooxidant-labeled β2 subunit of Escherichia coli class Ia RNR, we demonstrate photoinitiated oxidation of a tyrosine in an α2:β2 complex, which results in substrate turnover. Using site-directed mutations of the redox-active tyrosines at the subunit interface, Y356F(β) and Y731F(α), this oxidation is identified to be localized on Y356. The rate of Y356 oxidation depends on the presence of Y731 across the interface. This observation supports the proposal that unidirectional PCET across the Y356(β)-Y731(α)-Y730(α) triad is crucial to radical transport in RNR.

The class Ia ribonucleotide reductase (RNR) from Escherichia coli employs a free-radical mechanism, which involves bidirectional translocation of a radical equivalent or "hole" over a distance of ~35 Å from the stable diferric/tyrosyl-radical (Y122(•)) cofactor in the β subunit to cysteine 439 (C439) in the active site of the α subunit. This long-range, intersubunit electron transfer occurs by a multistep "hopping" mechanism via formation of transient amino acid radicals along a specific pathway and is thought to be conformationally gated and coupled to local proton transfers. Whereas constituent amino acids of the hopping pathway have been identified, details of the proton-transfer steps and conformational gating within the β sununit have remained obscure; specific proton couples have been proposed, but no direct evidence has been provided. In the key first step, the reduction of Y122(•) by the first residue in the hopping pathway, a water ligand to Fe1 of the diferric cluster was suggested to donate a proton to yield the neutral Y122. Here we show that forward radical translocation is associated with perturbation of the Mössbauer spectrum of the diferric cluster, especially the quadrupole doublet associated with Fe1. Density functional theory (DFT) calculations verify the consistency of the experimentally observed perturbation with that expected for deprotonation of the Fe1-coordinated water ligand. The results thus provide the first evidence that the diiron cluster of this prototypical class Ia RNR functions not only in its well-known role as generator of the enzyme's essential Y122(•), but also directly in catalysis.

Ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates (dNDPs). The Escherichia coli class Ia RNR uses a mechanism of radical propagation by which a cysteine in the active site of the RNR large (α2) subunit is transiently oxidized by a stable tyrosyl radical (Y•) in the RNR small (β2) subunit over a 35-Å pathway of redox-active amino acids: Y122• ↔ [W48?] ↔ Y356 in β2 to Y731 ↔ Y730 ↔ C439 in α2. When 3-aminotyrosine (NH2Y) is incorporated in place of Y730, a long-lived NH2Y730• is generated in α2 in the presence of wild-type (wt)-β2, substrate, and effector. This radical intermediate is chemically and kinetically competent to generate dNDPs. Herein, evidence is presented that NH2Y730• induces formation of a kinetically stable α2β2 complex. Under conditions that generate NH2Y730•, binding between Y730NH2Y-α2 and wt-β2 is 25-fold tighter (Kd = 7 nM) than for wt-α2

Escherichia coli class I ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two subunits: α2 and β2. β2 contains a stable di-iron tyrosyl radical (Y(122)(•)) cofactor required to generate a thiyl radical (C(439)(•)) in α2 over a distance of 35 Å, which in turn initiates the chemistry of the reduction process. The radical transfer process is proposed to occur by proton-coupled electron transfer (PCET) via a specific pathway: Y(122) ⇆ W(48)[?] ⇆ Y(356) in β2, across the subunit interface to Y(731) ⇆ Y(730) ⇆ C(439) in α2. Within α2 a colinear PCET model has been proposed. To obtain evidence for this model, 3-amino tyrosine (NH(2)Y) replaced Y(730) in α2, and this mutant was incubated with β2, cytidine 5'-diphosphate, and adenosine 5'-triphosphate to generate a NH(2)Y(730)(•) in D(2)O. [(2)H]-Electron-nuclear double resonance (ENDOR) spectra at 94 GHz of this intermediate were obtained, and together with DFT models of α2 and quantum chemical calculations allowed assignment of the prominent ENDOR features to two hydrogen bonds likely associated with C(439) and Y(731). A third proton was assigned to a water molecule in close proximity (2.2 Å O-H···O distance) to residue 730. The calculations also suggest that the unusual g-values measured for NH(2)Y(730)(•) are consistent with the combined effect of the hydrogen bonds to Cys(439) and Tyr(731), both nearly perpendicular to the ring plane of NH(2)Y(730.) The results provide the first experimental evidence for the hydrogen-bond network between the pathway residues in α2 of the active RNR complex, for which no structural data are available.

Incorporation of 2,3,6-trifluorotyrosine (F(3)Y) and a rhenium bipyridine ([Re]) photooxidant into a peptide corresponding to the C-terminus of the β protein (βC19) of Escherichia coli ribonucleotide reductase (RNR) allows for the temporal monitoring of radical transport into the α2 subunit of RNR. Injection of the photogenerated F(3)Y radical from the [Re]-F(3)Y-βC19 peptide into the surface accessible Y731 of the α2 subunit is only possible when the second Y730 is present. With the Y-Y established, radical transport occurs with a rate constant of 3 × 10(5) s(-1). Point mutations that disrupt the Y-Y dyad shut down radical transport. The ability to obviate radical transport by disrupting the hydrogen bonding network of the amino acids composing the colinear proton-coupled electron transfer pathway in α2 suggests a finely tuned evolutionary adaptation of RNR to control the transport of radicals in this enzyme., (© 2011 American Chemical Society)

Photochemical radical initiation is a powerful tool for studying radical initiation and transport in biology. Ribonucleotide reductases (RNRs), which catalyze the conversion of nucleotides to deoxynucleotides in all organisms, are an exemplar of radical mediated transformations in biology. Class Ia RNRs are composed of two subunits: α2 and β2. As a method to initiate radical formation photochemically within β2, a single surface-exposed cysteine of the β2 subunit of Escherichia coli Class Ia RNR has been labeled (98%) with a photooxidant ([Re ] = tricarbonyl(1,10-phenanthroline)(methylpyridyl)rhenium(I)). The labeling was achieved by incubation of S355C-β2 with the 4-(bromomethyl)pyridyl derivative of [Re] to yield the labeled species, [Re]-S355C-β2. Steady-state and time-resolved emission experiments reveal that the metal-to-ligand charge transfer (MLCT) excited-state (3)[Re ](∗) is not significantly perturbed after bioconjugation and is available as a phototrigger of tyrosine radical at position 356 in the β2 subunit; transient absorption spectroscopy reveals that the radical lives for microseconds. The work described herein provides a platform for photochemical radical initiation and study of proton-coupled electron transfer (PCET) in the β2 subunit of RNR, from which radical initiation and transport for this enzyme originates.

Essential for DNA biosynthesis and repair, ribonucleotide reductases (RNRs) convert ribonucleotides to deoxyribonucleotides via radical-based chemistry. Although long known that allosteric regulation of RNR activity is vital for cell health, the molecular basis of this regulation has been enigmatic, largely due to a lack of structural information about how the catalytic subunit (α(2)) and the radical-generation subunit (β(2)) interact. Here we present the first structure of a complex between α(2) and β(2) subunits for the prototypic RNR from Escherichia coli. Using four techniques (small-angle X-ray scattering, X-ray crystallography, electron microscopy, and analytical ultracentrifugation), we describe an unprecedented α(4)β(4) ring-like structure in the presence of the negative activity effector dATP and provide structural support for an active α(2)β(2) configuration. We demonstrate that, under physiological conditions, E. coli RNR exists as a mixture of transient α(2)β(2) and α(4)β(4) species whose distributions are modulated by allosteric effectors. We further show that this interconversion between α(2)β(2) and α(4)β(4) entails dramatic subunit rearrangements, providing a stunning molecular explanation for the allosteric regulation of RNR activity in E. coli.

Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides using a diferric tyrosyl radical (Y(122)(•)) 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 (Y(122)(•) ⇆ [W(48)?] ⇆ Y(356) within β to Y(731) ⇆ Y(730) ⇆ C(439) within α). Previously, we reported that a β2 mutant with 3-nitrotyrosyl radical (NO(2)Y(•); 1.2 radicals/β2) in place of Y(122)(•) 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 Y(356) in β2. We now report three independent methods that establish that Y(356) is the predominant location (85-90%) of the radical, with the remaining 10-15% delocalized onto Y(731) and Y(730) 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 NO(2)Y(122)(•)-Y(356)(•), NO(2)Y(122)(•)-NO(2)Y(122)(•), and NO(2)Y(122)(•)-Y(731(730))(•), respectively. Radical distribution in α2 was supported by RFQ electron paramagnetic resonance (EPR) studies using Y(731)(3,5-F(2)Y) or Y(730)(3,5-F(2)Y)-α2, which revealed F(2)Y(•), studies using globally incorporated [β-(2)H(2)]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.

Tyrosyl radicals (Y·s) are prevalent in biological catalysis and are formed under physiological conditions by the coupled loss of both a proton and an electron. Fluorotyrosines (F(n)Ys, n = 1-4) are promising tools for studying the mechanism of Y· formation and reactivity, as their pK(a) values and peak potentials span four units and 300 mV, respectively, between pH 6 and 10. In this manuscript, we present the directed evolution of aminoacyl-tRNA synthetases (aaRSs) for 2,3,5-trifluorotyrosine (2,3,5-F(3)Y) and demonstrate their ability to charge an orthogonal tRNA with a series of F(n)Ys while maintaining high specificity over Y. An evolved aaRS is then used to incorporate F(n)Ys site-specifically into the two subunits (α2 and β2) of Escherichia coli class Ia ribonucleotide reductase (RNR), an enzyme that employs stable and transient Y·s to mediate long-range, reversible radical hopping during catalysis. Each of four conserved Ys in RNR is replaced with F(n)Y(s), and the resulting proteins are isolated in good yields. F(n)Ys incorporated at position 122 of β2, the site of a stable Y· in wild-type RNR, generate long-lived F(n)Y·s that are characterized by electron paramagnetic resonance (EPR) spectroscopy. Furthermore, we demonstrate that the radical pathway in the mutant Y(122)(2,3,5)F(3)Y-β2 is energetically and/or conformationally modulated in such a way that the enzyme retains its activity but a new on-pathway Y· can accumulate. The distinct EPR properties of the 2,3,5-F(3)Y· facilitate spectral subtractions that make detection and identification of new Y·s straightforward.

Escherichia coli ribonucleotide reductase is an α2β2 complex and catalyzes the conversion of nucleoside 5'-diphosphates (NDPs) to 2'-deoxynucleotides (dNDPs). The reaction is initiated by the transient oxidation of an active-site cysteine (C(439)) in α2 by a stable diferric tyrosyl radical (Y(122)•) cofactor in β2. This oxidation occurs by a mechanism of long-range proton-coupled electron transfer (PCET) over 35 Å through a specific pathway of residues: Y(122)•→ W(48)→ Y(356) in β2 to Y(731)→ Y(730)→ C(439) in α2. To study the details of this process, 3-aminotyrosine (NH(2)Y) has been site-specifically incorporated in place of Y(356) of β. The resulting protein, Y(356)NH(2)Y-β2, and the previously generated proteins Y(731)NH(2)Y-α2 and Y(730)NH(2)Y-α2 (NH(2)Y-RNRs) are shown to catalyze dNDP production in the presence of the second subunit, substrate (S), and allosteric effector (E) with turnover numbers of 0.2-0.7 s(-1). Evidence acquired by three different methods indicates that the catalytic activity is inherent to NH(2)Y-RNRs and not the result of copurifying wt enzyme. The kinetics of formation of 3-aminotyrosyl radical (NH(2)Y•) at position 356, 731, and 730 have been measured with all S/E pairs. In all cases, NH(2)Y• formation is biphasic (k(fast) of 9-46 s(-1) and k(slow) of 1.5-5.0 s(-1)) and kinetically competent to be an intermediate in nucleotide reduction. The slow phase is proposed to report on the conformational gating of NH(2)Y• formation, while the k(cat) of ~0.5 s(-1) is proposed to be associated with rate-limiting oxidation by NH(2)Y• of the subsequent amino acid on the pathway during forward PCET. The X-ray crystal structures of Y(730)NH(2)Y-α2 and Y(731)NH(2)Y-α2 have been solved and indicate minimal structural changes relative to wt-α2. From the data, a kinetic model for PCET along the radical propagation pathway is proposed.

Escherichia coli class Ib ribonucleotide reductase (RNR) converts nucleoside 5'-diphosphates to deoxynucleoside 5'-diphosphates in iron-limited and oxidative stress conditions. We have recently demonstrated in vitro that this RNR is active with both diferric-tyrosyl radical (Fe(III)(2)-Y(•)) and dimanganese(III)-Y(•) (Mn(III)(2)-Y(•)) cofactors in the β2 subunit, NrdF [Cotruvo, J. A., Jr., and Stubbe, J. (2010) Biochemistry 49, 1297-1309]. Here we demonstrate, by purification of this protein from its endogenous levels in an E. coli strain deficient in its five known iron uptake pathways and grown under iron-limited conditions, that the Mn(III)(2)-Y(•) cofactor is assembled in vivo. This is the first definitive determination of the active cofactor of a class Ib RNR purified from its native organism without overexpression. From 88 g of cell paste, 150 μg of NrdF was isolated with ∼95% purity, with 0.2 Y(•)/β2, 0.9 Mn/β2, and a specific activity of 720 nmol min(-1) mg(-1). Under these conditions, the class Ib RNR is the primary active RNR in the cell. Our results strongly suggest that E. coli NrdF is an obligate manganese protein in vivo and that the Mn(III)(2)-Y(•) cofactor assembly pathway we have identified in vitro involving the flavodoxin-like protein NrdI, present inside the cell at catalytic levels, is operative in vivo.

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 (C(439)), 35 Å removed, is in β2. The oxidation is proposed to involve a hopping mechanism through aromatic amino acids (Y(122) → W(48) → Y(356) in β2 to Y(731) → Y(730) → C(439) in α2) and reversible proton-coupled electron transfer (PCET). Recently, 2,3,5-F(3)Y (F(3)Y) was site-specifically incorporated in place of Y(356) in β2 and 3-NH(2)Y (NH(2)Y) in place of Y(731) and Y(730) in α2. A pH-rate profile with F(3)Y(356)-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 F(3)Y oxidation, by residues adjacent to it in the pathway, is responsible for this change. Studies with NH(2)Y(731(730))-α2, β2, CDP, and ATP resulted in detection of NH(2)Y radical (NH(2)Y(•)) intermediates capable of dNDP formation. In this study, the reaction of F(3)Y(356)-β2, 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 F(3)Y(356)-β2, CDP, and ATP has also been examined with NH(2)Y(731)-α2 (or NH(2)Y(730)-α2) by SF kinetics from pH 6.5 to 9.2 and exhibited rate constants for NH(2)Y(•) 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.

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

Escherichia coli ribonucleotide reductase (RNR), an alpha2beta2 complex, catalyzes the conversion of nucleoside 5'-diphosphate substrates (S) to 2'-deoxynucleoside 5'-diphosphates. alpha2 houses the active site for nucleotide reduction and the binding sites for allosteric effectors (E). beta2 contains the essential diferric tyrosyl radical (Y(122)(*)) cofactor which, in the presence of S and E, oxidizes C(439) in alpha to a thiyl radical, C(439)(*), to initiate nucleotide reduction. This oxidation occurs over 35 A and is proposed to involve a specific pathway: Y(122)(*) --> W(48) --> Y(356) in beta2 to Y(731) --> Y(730) --> C(439) in alpha2. 3-Aminotyrosine (NH(2)Y) has been site-specifically incorporated at residues 730 and 731, and formation of the aminotyrosyl radical (NH(2)Y(*)) has been examined by stopped-flow (SF) UV-vis and EPR spectroscopies. To examine the pathway dependence of radical propagation, the double mutant complexes Y(356)F-beta2:Y(731)NH(2)Y-alpha2, Y(356)F-beta2:Y(730)NH(2)Y-alpha2, and wt-beta2:Y(731)F/Y(730)NH(2)Y-alpha2, in which the nonoxidizable F acts as a pathway block, were studied by SF and EPR spectroscopies. In all cases, no NH(2)Y(*) was detected. To study off-pathway oxidation, Y(413), located 5 A from Y(730) and Y(731) but not implicated in long-range oxidation, was examined. Evidence for NH(2)Y(413)(*) was sought in three complexes: wt-beta2:Y(413)NH(2)Y-alpha2 (a), wt-beta2:Y(731)F/Y(413)NH(2)Y-alpha2 (b), and Y(356)F-beta2:Y(413)NH(2)Y-alpha2 (c). With (a), NH(2)Y(*) was formed with a rate constant that was 25-30% and an amplitude that was 25% of that observed for its formation at residues 731 and 730. With (b), the rate constant for NH(2)Y(*) formation was 0.2-0.3% of that observed at 731 and 730, and with (c), no NH(2)Y(*) was observed. These studies suggest the evolution of an optimized pathway of conserved Ys in the oxidation of C(439).

E. coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, a process that requires long-range radical transfer over 35 A from a tyrosyl radical (Y(122)*) within the beta2 subunit to a cysteine residue (C(439)) within the alpha2 subunit. The radical transfer step is proposed to occur by proton-coupled electron transfer via a specific pathway consisting of Y(122) --> W(48) --> Y(356) in beta2, across the subunit interface to Y(731) --> Y(730) --> C(439) in alpha2. Using the suppressor tRNA/aminoacyl-tRNA synthetase (RS) methodology, 3-aminotyrosine has been incorporated into position 730 in alpha2. Incubation of this mutant with beta2, substrate, and allosteric effector resulted in loss of the Y(122)* and formation of a new radical, previously proposed to be a 3-aminotyrosyl radical (NH(2)Y*). In the current study [(15)N]- and [(14)N]-NH(2)Y(730)* have been generated in H(2)O and D(2)O and characterized by continuous wave 9 GHz EPR and pulsed EPR spectroscopies at 9, 94, and 180 GHz. The data give insight into the electronic and molecular structure of NH(2)Y(730)*. The g tensor (g(x) = 2.0052, g(y) = 2.0042, g(z) = 2.0022), the orientation of the beta-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 NH(2) moiety are consistent with an intramolecular hydrogen bond within NH(2)Y(730)*. This analysis is an essential first step in using the detailed structure of NH(2)Y(730)* to formulate a model for a PCET mechanism within alpha2 and for use of NH(2)Y in other systems where transient Y*s participate in catalysis.

The Escherichia 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: alpha and beta. Incubation of the E441Q-alpha 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'-(2)H], [2'-(2)H], [4'-(2)H], [5'-(2)H], [U-(13)C, (15)N], [U-(15)N], and [5,6 -(2)H] CDP and [beta-(2)H] cysteine-alpha were synthesized and incubated with E441Q-alpha2beta2 and TTP. The nucleotide radical was examined by 9 GHz and 140 GHz pulsed EPR spectroscopy and 35 GHz ENDOR spectroscopy. Substitution of (2)H 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-III/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.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides, providing the monomeric precursors required for DNA replication and repair. Escherichia coli RNR is a 1:1 complex of two homodimeric subunits, alpha2 and beta2. The interactions between alpha2 and beta2 are thought to be largely associated with the C-terminal 20 amino acids (residues 356-375) of beta2. To study subunit interactions, a single reactive cysteine has been introduced into each of 15 positions along the C-terminal tail of beta2. Each cysteine has been modified with the photo-cross-linker benzophenone (BP) and the environmentally sensitive fluorophore dimethylaminonaphthalene (DAN). Each construct has been purified to homogeneity and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrospray ionization mass spectrometry (ESI-MS). Each BP-beta2 has been incubated with 1 equiv of alpha2 and photolyzed, and the results have been analyzed quantitatively by SDS-PAGE. Each DAN-beta2 was incubated with a 50-fold excess of alpha2, and the emission maximum and intensity were measured. A comparison of the results from the two sets of probes reveals that sites with the most extensive cross-linking are also associated with the greatest changes in fluorescence. Titration of four different DAN-beta2 variants (351, 356, 365, and 367) with alpha2 gave a K(d) approximately 0.4 microM for subunit interaction. Disruption of the interaction of the alpha2-DAN-beta2 complex is accompanied by a decrease in fluorescence intensity and can serve as a high-throughput screen for inhibitors of subunit interactions.

Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside 5'-diphosphates to deoxynucleoside 5'-diphosphates and is a 1:1 complex of two homodimeric subunits: alpha2 and beta2. As a first step towards mapping the subunit interface, beta2 (V365C) was labeled with [(14)C]-benzophenone (BP) iodoacetamide. The resulting [(14)C]-BP-beta2 (V365C) was complexed with alpha2 and irradiated at 365nm for 30min at 4 degrees C. The cross-linked mixture was purified by anion exchange chromatography and digested with trypsin. The peptides were purified by reverse phase chromatography, identified by scintillation counting and analyzed by Edman sequencing. Three [(14)C]-labeled peptides were identified: two contained a peptide in beta to which the BP was attached. The third contained the same beta peptide and a peptide in alpha found in its alphaD helix. These results provide direct support for the proposed docking model of alpha2beta2.

Ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides and is essential in all organisms. Class I RNRs consist of two homodimeric subunits: alpha2 and beta2. The alpha subunit contains the site of nucleotide reduction, and the beta 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 (alpha) and NrdB (beta), is expressed under normal aerobic growth conditions. The class Ib RNR, composed of NrdE (alpha) and NrdF (beta), 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 Ib RNR. It possesses redox properties unprecedented for a flavodoxin (E(ox/sq) = -264 +/- 17 mV and E(sq/hq) = -255 +/- 17 mV) 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.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. The Escherichia coli class Ia RNR is composed of alpha and beta subunits that form an alpha 2beta 2 active complex. beta contains the diferric tyrosyl radical (Y (*)) cofactor that is essential for the reduction process that occurs on alpha. [Y (*)] in vitro is proportional to RNR activity, and its regulation in vivo potentially represents a mechanism for controlling RNR activity. To examine this thesis, N- and C-terminal StrepII-tagged beta under the control of an l-arabinose promoter were constructed. Using these constructs and with [ l-arabinose] varying from 0 to 0.5 mM in the growth medium, [beta] could be varied from 4 to 3300 microM. [Y (*)] in vivo and on affinity-purified Strep-beta in vitro was determined by EPR spectroscopy and Western analysis. In both cases, there was 0.1-0.3 Y (*) radical per beta. To determine if the substoichiometric Y (*) level was associated with apo beta or diferric beta, titrations of crude cell extracts from these growths were carried out with reduced YfaE, a 2Fe2S ferredoxin involved in cofactor maintenance and assembly. Each titration, followed by addition of O 2 to assemble the cofactor and EPR analysis to quantitate Y (*), revealed that beta is completely loaded with a diferric cluster even when its concentration in vivo is 244 microM. These titrations, furthermore, resulted in 1 Y (*) radical per beta, the highest levels reported. Whole cell Mössbauer analysis on cells induced with 0.5 mM arabinose supports high iron loading in beta. These results suggest that modulation of the level of Y (*) in vivo in E. coli is a mechanism of regulating RNR activity.

Escherichia coli ribonucleotide reductase (RNR) catalyzes the reduction of nucleotides to 2'-deoxynucleotides. The active enzyme is a 1:1 complex of two homodimeric subunits, alpha2 and beta2. The alpha2 is the site of nucleotide reduction, and beta2 harbors a diferric tyrosyl radical (Y122*) cofactor. Turnover requires formation of a cysteinyl radical (C439*) in the active site of alpha2 at the expense of the Y122* in beta2. A docking model for the alpha2beta2 interaction and a pathway for radical transfer from beta2 to alpha2 have been proposed. This pathway contains three Ys: Y356 in beta2 and Y731/Y730 in alpha2. We have previously incorporated 3-hydroxytyrosine and 3-aminotyrosine into these residues and showed that they act as radical traps. In this study, we use these alpha2/beta2 variants and PELDOR spectroscopy to measure the distance between the Y122* in one alphabeta pair and the newly formed radical in the second alphabeta pair. The results yield distances that are similar to those predicted by the docking model for radical transfer. Further, they support a long-range radical initiation process for C439* generation and provide a structural constraint for residue Y356, which is thermally labile in all beta2 structures solved to date.

E. coli ribonucleotide reductase (RNR) catalyzes the production of deoxynucleotides using complex radical chemistry. Active RNR is composed of a 1:1 complex of two subunits: alpha2 and beta2. Alpha2 binds nucleoside diphosphate substrates and deoxynucleotide/ATP allosteric effectors and is the site of nucleotide reduction. Beta2 contains the stable diiron tyrosyl radical (Y122.) cofactor that initiates deoxynucleotide formation. This process is proposed to involve reversible radical transfer over >35 A between the Y122 in beta2 and C439 in the active site of alpha2. A docking model of alpha2beta2, based on structures of the individual subunits, suggests that radical initiation involves a pathway of transient, aromatic amino acid radical intermediates, including Y730 and Y731 in alpha2. In this study the function of residues Y730 and Y731 is investigated by their site-specific replacement with 3-aminotyrosine (NH2Y). Using the in vivo suppressor tRNA/aminoacyl-tRNA synthetase method, Y730NH2Y-alpha2 and Y731NH2Y-alpha2 have been generated with high fidelity in yields of 4-6 mg/g of cell paste. These mutants have been examined by stopped flow UV-vis and EPR spectroscopies in the presence of beta2, CDP, and ATP. The results reveal formation of an NH2Y radical (NH2Y730. or NH2Y731.) in a kinetically competent fashion. Activity assays demonstrate that both NH2Y-alpha2s make deoxynucleotides. These results show that the NH2Y. can oxidize C439 suggesting a hydrogen atom transfer mechanism for the radical propagation pathway within alpha2. The observed NH2Y. may constitute the first detection of an amino acid radical intermediate in the proposed radical propagation pathway during turnover.

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: alpha and beta. beta contains the diferric-tyrosyl radical (Y*) cofactor essential for the reduction process. In vivo, the mechanism of Y* regeneration from the diferric-beta2 (met-beta2) or apo-beta2 is still unclear. Y* regenerations from met-beta2 and apo-beta2 have been designated the maintenance and biosynthetic pathways, respectively. To understand these two pathways, 181 genomes that contain nrdAnrdB (genes encoding alpha and beta) 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-beta2 with [2Fe2S]1+-YfaE anaerobically results in the formation of an equilibrium mixture of diferrous-beta2 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-beta2 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 beta. 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.

The Escherichia coli ribonucleotide reductase (RNR), composed of two subunits (R1 and R2), catalyzes the conversion of nucleotides to deoxynucleotides. Substrate reduction requires that a tyrosyl radical (Y(122)*) in R2 generate a transient cysteinyl radical (C(439)*) in R1 through a pathway thought to involve amino acid radical intermediates [Y(122)* --> W(48) --> Y(356) within R2 to Y(731) --> Y(730) --> C(439) within R1]. To study this radical propagation process, we have synthesized R2 semisynthetically using intein technology and replaced Y(356) with a variety of fluorinated tyrosine analogues (2,3-F(2)Y, 3,5-F(2)Y, 2,3,5-F(3)Y, 2,3,6-F(3)Y, and F(4)Y) that have been described and characterized in the accompanying paper. These fluorinated tyrosine derivatives have potentials that vary from -50 to +270 mV relative to tyrosine over the accessible pH range for RNR and pK(a)s that range from 5.6 to 7.8. The pH rate profiles of deoxynucleotide production by these F(n)()Y(356)-R2s are reported. The results suggest that the rate-determining step can be changed from a physical step to the radical propagation step by altering the reduction potential of Y(356)* using these analogues. As the difference in potential of the F(n)()Y* relative to Y* becomes >80 mV, the activity of RNR becomes inhibited, and by 200 mV, RNR activity is no longer detectable. These studies support the model that Y(356) is a redox-active amino acid on the radical-propagation pathway. On the basis of our previous studies with 3-NO(2)Y(356)-R2, we assume that 2,3,5-F(3)Y(356), 2,3,6-F(3)Y(356), and F(4)Y(356)-R2s are all deprotonated at pH > 7.5. We show that they all efficiently initiate nucleotide reduction. If this assumption is correct, then a hydrogen-bonding pathway between W(48) and Y(356) of R2 and Y(731) of R1 does not play a central role in triggering radical initiation nor is hydrogen-atom transfer between these residues obligatory for radical propagation.

The class I E. coli ribonucleotide reductase, composed of homodimers of R1 and R2, catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates. The reduction process involves the tyrosyl radical on R2 that generates a transient thiyl radical on R1 over a proposed distance of 35 A. A mechanism-based inhibitor, 2'-azido-2'-deoxyuridine-5'-diphosphate, that reduces the tyrosyl radical on R2 and forms a nitrogen-centered radical on R1 has provided a method to measure the diagonal distance between the two subunits. PELDOR and DQC paramagnetic resonance methods give rise to a distance of 48 A, similar to that calculated from a docking model of the R1 and R2 structures.

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 (N(3)UDP), have provided much insight into the reduction mechanism. N(3)UDP 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. N(2) 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'-[(17)O]-N(3)UDP 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 [(17)O] hyperfine tensor. DFT calculations were employed to determine which structures are in best agreement with the simulated hyperfine 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.

Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates (NDPs) to deoxynucleoside diphosphates (dNDPs). This RNR is composed of two homodimeric subunits: R1 and R2. R1 binds the NDPs in the active site, and R2 harbors the essential di-iron tyrosyl radical (Y*) cofactor. In this paper, we used PELDOR, a method that detects weak electron-electron dipolar coupling, to make the first direct measurement of the distance between the two Y*'s on each monomer of R2. In the crystal structure of R2, the Y*'s are reduced to tyrosines, and consequently R2 is inactive. In R2, where the Y*'s assume a well-defined geometry with respect to the protein backbone, the PELDOR method allows measurement of a distance of 33.1 +/- 0.2 A that compares favorably to the distance (32.4 A) between the center of mass of the spin density distribution of each Y* on each R2 monomer from the structure. The experiments provide the first direct experimental evidence for two Y*'s in a single R2 in solution.

E. coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates (NDPs) to dNDPs and is composed of two homodimeric subunits: R1 and R2. R1 binds NDPs and contains binding sites for allosteric effectors that control substrate specificity and turnover rate. R2 contains a diiron-tyrosyl radical (Y(*)) cofactor that initiates nucleotide reduction. Pre-steady-state experiments with wild type R1 or C754S/C759S-R1 and R2 were carried out to determine which step(s) are rate-limiting and whether both active sites of R1 can catalyze nucleotide reduction. Rapid chemical quench experiments monitoring dCDP formation gave k(obs) of 9 +/- 4 s(-1) with an amplitude of 1.7 +/- 0.4 equiv. This amplitude, generated in experiments with pre-reduced R1 (3 or 15 microM) in the absence of reductant, indicates that both monomers of R1 are active. Stopped-flow UV-vis spectroscopy monitoring the concentration of the Y(*) failed to reveal any changes from 2 ms to seconds under similar conditions. These pre-steady-state experiments, in conjunction with the steady-state turnover numbers for dCDP formation of 2-14 s(-1) at RNR concentrations of 0.05-0.4 microM (typical assay conditions), reveal that the rate-determining step is a physical step prior to rapid nucleotide reduction and rapid tyrosine reoxidation to Y(*). Steady-state experiments conducted at RNR concentrations of 3 and 15 microM, typical of pre-steady-state conditions, suggest that, in addition to the slow conformational change(s) prior to chemistry, re-reduction of the active site disulfide to dithiol or a conformational change accompanying this process can also be rate-limiting.

Ribonucleotide reductases (RNRs) catalyze the de novo conversion of nucleotides to deoxynucleotides in all organisms, controlling their relative ratios and abundance. In doing so, they play an important role in fidelity of DNA replication and repair. RNRs' central role in nucleic acid metabolism has resulted in five therapeutics that inhibit human RNRs. In this review, we discuss the structural, dynamic, and mechanistic aspects of RNR activity and regulation, primarily for the human and Escherichia coli class Ia enzymes. The unusual radical-based organic chemistry of nucleotide reduction, the inorganic chemistry of the essential metallo-cofactor biosynthesis/maintenance, the transport of a radical over a long distance, and the dynamics of subunit interactions all present distinct entry points toward RNR inhibition that are relevant for drug discovery. We describe the current mechanistic understanding of small molecules that target different elements of RNR function, including downstream pathways that lead to cell cytotoxicity. We conclude by summarizing novel and emergent RNR targeting motifs for cancer and antibiotic therapeutics.

Chris Raetz passed away on August 16,2011, still at the height of his productive years. His seminal contributions to biomedical research were in the genetics, biochemistry, and structural biology of phospholipid and lipid A biosynthesis in Escherichia coli and other gramnegative bacteria. He defined the catalytic properties and structures of many of the enzymes responsible for the "Raetz pathway for lipid A biosynthesis." His deep understanding of chemistry, coupled with knowledge of medicine, biochemistry, genetics, and structural biology, formed the underpin-nings for his contributions to the lipid field. He displayed an intense passion for science and a broad interest that came from a strong commitment to curiositydriven research, a commitment he imparted to his mentees and colleagues. What follows is a testament to both Chris's science and humanity from his friends and colleagues. [ABSTRACT FROM AUTHOR]