Ribonucleotide reductases (RNR) catalyze the reduction of nucleotides to deoxynucleotides through a mechanism involving an essential cysteine based thiyl radical. In the E. coli class 1a RNR the thiyl radical (C439•) is a transient species generated by radical transfer (RT) from a stable diferric-tyrosyl radical cofactor located >35 Å away across the α2:β2 subunit interface. RT is facilitated by sequential proton-coupled electron transfer (PCET) steps along a pathway of redox active amino acids (Y122β ↔ [W48β?] ↔ Y356β ↔ Y731α ↔ Y730α ↔ C439α). The mutant R411A(α) disrupts the H-bonding environment and conformation of Y731, ostensibly breaking the RT pathway in α2. However, the R411A protein retains significant enzymatic activity, suggesting Y731 is conformationally dynamic on the time scale of turnover. Installation of the radical trap 3-amino tyrosine (NH2Y) by amber codon suppression at positions Y731 or Y730 and investigation of the NH2Y• trapped state in the active α2:β2 complex by HYSCORE spectroscopy validate that the perturbed conformation of Y731 in R411A-α2 is dynamic, reforming the H-bond between Y731 and Y730 to allow RT to propagate to Y730. Kinetic studies facilitated by photochemical radical generation reveal that Y731 changes conformation on the ns−μs time scale, significantly faster than the enzymatic kcat. Furthermore, the kinetics of RT across the subunit interface were directly assessed for the first time, demonstrating conformationally dependent RT rates that increase from 0.6 to 1.6 × 104 s–1 when comparing wild type to R411A-α2, respectively. These results illustrate the role of conformational flexibility in modulating RT kinetics by targeting the PCET pathway of radical transport.

Escherichia coli class Ia ribonucleotide reductase (RNR) is composed of two subunits that form an active α2β2 complex. The nucleoside diphosphate substrates (NDP) are reduced in α2, 35 Å from the essential diferric-tyrosyl radical (Y122•) cofactor in β2. The Y122•-mediated oxidation of C439 in α2 occurs by a pathway (Y122 ⇆ [W48] ⇆ Y356 in β2 to Y731 ⇆ Y730 ⇆ C439 in α2) across the α/β interface. The absence of an α2β2 structure precludes insight into the location of Y356 and Y731 at the subunit interface. The proximity in the primary sequence of the conserved E350 to Y356 in β2 suggested its importance in catalysis and/or conformational gating. To study its function, pH–rate profiles of wild-type β2/α2 and mutants in which 3,5-difluorotyrosine (F2Y) replaces residue 356, 731, or both are reported in the presence of E350 or E350X (X = A, D, or Q) mutants. With E350, activity is maintained at the pH extremes, suggesting that protonated and deprotonated states of F2Y356 and F2Y731 are active and that radical transport (RT) can occur across the interface by proton-coupled electron transfer at low pH or electron transfer at high pH. With E350X mutants, all RNRs were inactive, suggesting that E350 could be a proton acceptor during oxidation of the interface Ys. To determine if E350 plays a role in conformational gating, the strong oxidants, NO2Y122•-β2 and 2,3,5-F3Y122•-β2, were reacted with α2, CDP, and ATP in E350 and E350X backgrounds and the reactions were monitored for pathway radicals by rapid freeze-quench electron paramagnetic resonance spectroscopy. Pathway radicals are generated only when E350 is present, supporting its essential role in gating the conformational change(s) that initiates RT and masking its role as a proton acceptor.

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, Fe2+, and O2 to produce ∼1 Y·/β2 and ∼3 Fe3+/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 19F 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.

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides in all living organisms. The catalytic cycle of E. coli RNR involves a long-range proton-coupled electron transfer (PCET) from a tyrosyl radical (Y122˙) in subunit β2 to a cysteine (C439) in the active site of subunit α2, which subsequently initiates nucleotide reduction. This oxidation occurs over 35 Å and involves a specific pathway of redox active amino acids (Y122 ↔ [W48?] ↔ Y356 in β2 to Y731 ↔ Y730 ↔ C439 in α2). The mechanisms of the PCET steps at the interface of the α2β2 complex remain puzzling due to a lack of structural information for this region. Recently, DFT calculations on the 3-aminotyrosyl radical (NH2Y731˙)-α2 trapped by incubation of NH2Y731-α2/β2/CDP(substrate)/ATP(allosteric effector) suggested that R411-α2, a residue close to the α2β2 interface, interacts with NH2Y731˙ and accounts in part for its perturbed EPR parameters. To examine its role, we further modified NH2Y731-α2 with a R411A substitution. NH2Y731˙/R411A generated upon incubation of NH2Y731/R411A-α2/β2/CDP/ATP was investigated using multi-frequency (34, 94 and 263 GHz) EPR, 34 GHz pulsed electron–electron double resonance (PELDOR) and electron–nuclear double resonance (ENDOR) spectroscopies. The data indicate a large conformational change in NH2Y731˙/R411A relative to the NH2Y731˙ single mutant. Particularly, the inter-spin distance from NH2Y731˙/R411A in one αβ pair to Y122˙ in a second αβ pair decreases by 3 Å in the presence of the R411A mutation. This is the first experimental evidence for the flexibility of pathway residue Y731-α2 in an α2β2 complex and suggests a role for R411 in the stacked Y731/Y730 conformation involved in collinear PCET. Furthermore, NH2Y731˙/R411A serves as a probe of the PCET process across the subunit interface.

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) × 105 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.

Escherichia coli class Ia ribonucleotide reductase is composed of two subunits (α and β), which form an α2β2 complex that catalyzes the conversion of nucleoside 5′-diphosphates to deoxynucleotides (dNDPs). β2 contains the essential tyrosyl radical (Y122•) that generates a thiyl radical (C439•) in α2 where dNDPs are made. This oxidation occurs over 35 Å through a pathway of amino acid radical intermediates (Y122 → [W48] → Y356 in β2 to Y731 → Y730 → C439 in α2). However, chemistry is preceded by a slow protein conformational change(s) that prevents observation of these intermediates. 2,3,5-Trifluorotyrosine site-specifically inserted at position 122 of β2 (F3Y•-β2) perturbs its conformation and the driving force for radical propagation, while maintaining catalytic activity (1.7 s–1). Rapid freeze–quench electron paramagnetic resonance spectroscopy and rapid chemical-quench analysis of the F3Y•-β2, α2, CDP, and ATP (effector) reaction show generation of 0.5 equiv of Y356• and 0.5 equiv of dCDP, both at 30 s–1. In the absence of an external reducing system, Y356• reduction occurs concomitant with F3Y reoxidation (0.4 s–1) and subsequent to oxidation of all α2s. In the presence of a reducing system, a burst of dCDP (0.4 equiv at 22 s–1) is observed prior to steady-state turnover (1.7 s–1). The [Y356•] does not change, consistent with rate-limiting F3Y reoxidation. The data support a mechanism where Y122• is reduced and reoxidized on each turnover and demonstrate for the first time the ability of a pathway radical in an active α2β2 complex to complete the catalytic cycle.

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 57Fe, 14,15N, 17O, and 1H electron nuclear double resonance (ENDOR) measurements combined with quantitative measurements of 17O and 1H 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–)FeIII–O–FeIV]: there is no second inorganic oxygenic bridge, neither oxo nor hydroxo. Geometric analysis of the 14N 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.

Proton-coupled electron transfer (PCET) is a fundamental mechanism important in a wide range of biological processes including the universal reaction catalysed by ribonucleotide reductases (RNRs) in making de novo, the building blocks required for DNA replication and repair. These enzymes catalyse the conversion of nucleoside diphosphates (NDPs) to deoxynucleoside diphosphates (dNDPs). In the class Ia RNRs, NDP reduction involves a tyrosyl radical mediated oxidation occurring over 35 Å across the interface of the two required subunits (β2 and α2) involving multiple PCET steps and the conserved tyrosine triad [Y356(β2)–Y731(α2)–Y730(α2)]. We report the synthesis of an active photochemical RNR (photoRNR) complex in which a Re(I)-tricarbonyl phenanthroline ([Re]) photooxidant is attached site-specifically to the Cys in the Y356C-(β2) subunit and an ionizable, 2,3,5-trifluorotyrosine (2,3,5-F3Y) is incorporated in place of Y731 in α2. This intersubunit PCET pathway is investigated by ns laser spectroscopy on [Re356]-β2:2,3,5-F3Y731-α2 in the presence of substrate, CDP, and effector, ATP. This experiment has allowed analysis of the photoinjection of a radical into α2 from β2 in the absence of the interfacial Y356 residue. The system is competent for light-dependent substrate turnover. Time-resolved emission experiments reveal an intimate dependence of the rate of radical injection on the protonation state at position Y731(α2), which in turn highlights the importance of a well-coordinated proton exit channel involving the key residues, Y356 and Y731, at the subunit interface.

Polyhydroxybutyrate (PHB) synthases (PhaCs) catalyze the conversion of 3-(R)-hydroxybutyryl CoA (HBCoA) to PHB, which is deposited as granules in the cytoplasm of microorganisms. The class I PhaC from Caulobacter crescentus (PhaCCc) is a highly soluble protein with a turnover number of 75 s–1 and no lag phase in coenzyme A (CoA) release. Studies with [1-14C]HBCoA and PhaCCc monitored by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and autoradiography reveal that the rate of elongation is much faster than the rate of initiation. Priming with the artificial primer [3H]sTCoA and monitoring for CoA release reveal a single CoA/PhaC, suggesting that the protein is uniformly loaded and that the elongation process could be studied. Reaction of sT-PhaCCc with [1-14C]HBCoA revealed that priming with sTCoA increased the uniformity of elongation, allowing distinct polymerization species to be observed by SDS–PAGE and autoradiography. However, in the absence of HBCoA, [3H]sT-PhaC unexpectedly generates [3H]sDCoA with a rate constant of 0.017 s–1. We propose that the [3H]sDCoA forms via attack of CoA on the oxoester of the [3H]sT-PhaC chain, leaving the synthase attached to a single HB unit. Comparison of the relative rate constants of thiolysis by CoA and elongation by PhaCCc, and the size of the PHB polymer generated in vivo, suggests a mechanism for chain termination and reinitiation.

Two subtypes of class III anaerobic ribonucleotide reductases (RNRs) studied so far couple the reduction of ribonucleotides to the oxidation of formate, or the oxidation of NADPH via thioredoxin and thioredoxin reductase. Certain methanogenic archaea contain a phylogenetically distinct third subtype of class III RNR, with distinct active-site residues. Here we report the cloning and recombinant expression of the Methanosarcina barkeri class III RNR and show that the electrons required for ribonucleotide reduction can be delivered by a [4Fe-4S] protein ferredoxin disulfide reductase, and a conserved thioredoxin-like protein NrdH present in the RNR operon. The diversity of class III RNRs reflects the diversity of electron carriers used in anaerobic metabolism.

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. 2H 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 [ReI] 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′-[2H]-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) × 104 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 [3H]-(kcat/KM) isotope effect of 2.3 with [3H]-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.

Polyhydroxybutyrate (PHB) synthases catalyze the polymerization of 3-(R)-hydroxybutyrate coenzyme A (HBCoA) to produce polyoxoesters of 1–2 MDa. A substrate analogue HBCH2CoA, in which the S in HBCoA is replaced with a CH2 group, was synthesized in 13 steps using a chemoenzymatic approach in a 7.5% overall yield. Kinetic studies reveal it is a competitive inhibitor of a class I and a class III PHB synthases, with Kis of 40 and 14 μM, respectively. To probe the elongation steps of the polymerization, HBCH2CoA was incubated with a synthase acylated with a [3H]-saturated trimer-CoA ([3H]-sTCoA). The products of the reaction were shown to be the methylene analogue of [3H]-sTCoA ([3H]-sT-CH2-CoA), saturated dimer-([3H]-sD-CO2H), and trimer-acid ([3H]-sT-CO2H), distinct from the expected methylene analogue of [3H]-saturated tetramer-CoA ([3H]-sTet-CH2-CoA). Detection of [3H]-sT-CH2-CoA and its slow rate of formation suggest that HBCH2CoA may be reporting on the termination and repriming process of the synthases, rather than elongation.

The class Ib ribonucleotide reductase (RNR) isolated from Bacillus subtilis was recently purified as a 1:1 ratio of NrdE (α) and NrdF (β) subunits and determined to have a dimanganic-tyrosyl radical (MnIII2-Y·) cofactor. The activity of this RNR and the one reconstituted from recombinantly expressed NrdE and reconstituted MnIII2-Y· NrdF using dithiothreitol as the reductant, however, was low (160 nmol min–1 mg–1). The apparent tight affinity between the two subunits, distinct from all class Ia RNRs, suggested that B. subtilis RNR might be the protein that yields to the elusive X-ray crystallographic characterization of an “active” RNR complex. We now report our efforts to optimize the activity of B. subtilis RNR by (1) isolation of NrdF with a homogeneous cofactor, and (2) identification and purification of the endogenous reductant(s). Goal one was achieved using anion exchange chromatography to separate apo-/mismetalated-NrdFs from MnIII2-Y· NrdF, yielding enzyme containing 4 Mn and 1 Y·/β2. Goal two was achieved by cloning, expressing, and purifying TrxA (thioredoxin), YosR (a glutaredoxin-like thioredoxin), and TrxB (thioredoxin reductase). The success of both goals increased the specific activity to ∼1250 nmol min–1 mg–1 using a 1:1 mixture of NrdE:MnIII2-Y· NrdF and either TrxA or YosR and TrxB. The quaternary structures of NrdE, NrdF, and NrdE:NrdF (1:1) were characterized by size exclusion chromatography and analytical ultracentrifugation. At physiological concentrations (∼1 μM), NrdE is a monomer (α) and MnIII2-Y· NrdF is a dimer (β2). A 1:1 mixture of NrdE:NrdF, however, is composed of a complex mixture of structures in contrast to expectations.

Eukaryotic ribonucleotide reductases (RNRs) require a diferric-tyrosyl radical (FeIII2-Y•) cofactor to produce deoxynucleotides essential for DNA replication and repair. This metallocofactor is an important target of RNR-based therapeutics, although mechanisms of in vivo cofactor assembly, inactivation, and reactivation are poorly understood. Here, we demonstrate that the conserved Fe-S protein–diflavin reductase complex, Dre2–Tah18, plays a critical role in RNR cofactor biosynthesis. Depletion of Dre2 affects both RNR gene transcription and mRNA turnover through the activation of the DNA-damage checkpoint and the Aft1/Aft2-controlled iron regulon. Under conditions of comparable RNR protein levels, cells with diminishing Dre2 have significantly reduced ability to make deoxynucleotides. Furthermore, the kinetics and levels of in vivo reconstitution of the RNR cofactor are severely impaired in two conditional tah18 mutants. Together, these findings provide insight into RNR cofactor formation and reveal a shared mechanism underlying assembly of the FeIII2-Y• cofactor in RNR and the Fe-S clusters in cytosolic and nuclear proteins.

The class III anaerobic ribonucleotide reductases (RNRs) studied to date couple the reduction of ribonucleotides to deoxynucleotides with the oxidation of formate to CO2. Here we report the cloning and heterologous expression of the Neisseria bacilliformis class III RNR and show that it can catalyze nucleotide reduction using the ubiquitous thioredoxin/thioredoxin reductase/NADPH system. We present a structural model based on a crystal structure of the homologous Thermotoga maritima class III RNR, showing its architecture and the position of conserved residues in the active site. Phylogenetic studies suggest that this form of class III RNR is present in bacteria and archaea that carry out diverse types of anaerobic metabolism.

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 [2H]-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 ∼105 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|wt-β2 and is cooperative. Stopped-flow fluorescence experiments establish that the dissociation rate constant for the Y730NH2Y-α2|wt-β2 interaction is ∼104-fold slower than for the wt subunits (∼60 s−1). EM and small-angle X-ray scattering studies indicate that the stabilized species is a compact globular α2β2, consistent with the structure predicted by Uhlin and Eklund’s docking model [Uhlin U, Eklund H (1994) Nature 370(6490):533–539]. These results present a structural and biochemical characterization of the active RNR complex “trapped” during turnover, and suggest that stabilization of the α2β2 state may be a regulatory mechanism for protecting the catalytic radical and ensuring the fidelity of its reactivity.

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.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (91 K)Download as PowerPoint slideHighlights► Expression/isolation of active human RNR-α from mammalian cells is developed ► Strategy unveils Clofarabine mechanism and in-cell-relevance of α-oligomerization ► Independent biochemical and structural methods document in-cell-assembled α-hexamers ► Drug-induced hexameric states have remarkable kinetic stability

How cells ensure correct metallation of a given protein and whether a degree of promiscuity in metal binding has evolved are largely unanswered questions. In a classic case, iron- and manganese-dependent superoxide dismutases (SODs) catalyze the disproportionation of superoxide using highly similar protein scaffolds and nearly identical active sites. However, most of these enzymes are active with only one metal, although both metals can bind in vitro and in vivo. Iron(II) and manganese(II) bind weakly to most proteins and possess similar coordination preferences. Their distinct redox properties suggest that they are unlikely to be interchangeable in biological systems except when they function in Lewis acid catalytic roles, yet recent work suggests this is not always the case. This review summarizes the diversity of ways in which iron and manganese are substituted in similar or identical protein frameworks. As models, we discuss (1) enzymes, such as epimerases, thought to use FeII as a Lewis acid under normal growth conditions but which switch to MnII under oxidative stress; (2) extradiol dioxygenases, which have been found to use both FeII and MnII, the redox role of which in catalysis remains to be elucidated; (3) SODs, which use redox chemistry and are generally metal-specific; and (4) the class I ribonucleotide reductases (RNRs), which have evolved unique biosynthetic pathways to control metallation. The primary focus is the class Ib RNRs, which can catalyze formation of a stable radical on a tyrosine residue in their β2 subunits using either a di-iron or a recently characterized dimanganese cofactor. The physiological roles of enzymes that can switch between iron and manganese cofactors are discussed, as are insights obtained from the studies of many groups regarding iron and manganese homeostasis and the divergent and convergent strategies organisms use for control of protein metallation. We propose that, in many of the systems discussed, “discrimination” between metals is not performed by the protein itself, but it is instead determined by the environment in which the protein is expressed.

Class I polyhydroxybutyrate (PHB) synthase (PhaC) from Ralstonia eutropha catalyzes the formation of PHB from (R)-3-hydroxybutyryl-CoA, ultimately resulting in the formation of insoluble granules. Previous mechanistic studies of R. eutropha PhaC, purified from Escherichia coli (PhaCEc), demonstrated that the polymer elongation rate is much faster than the initiation rate. In an effort to identify a factor(s) from the native organism that might prime the synthase and increase the rate of polymer initiation, an N-terminally Strep2-tagged phaC (Strep2-PhaCRe) was constructed and integrated into the R. eutropha genome in place of wild-type phaC. Strep2-PhaCRe was expressed and purified by affinity chromatography from R. eutropha grown in nutrient-rich TSB medium for 4 h (peak production PHB, 15% cell dry weight) and 24 h (PHB, 2% cell dry weight). Analysis of the purified PhaC by size exclusion chromatography, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and gel permeation chromatography revealed that it unexpectedly copurified with the phasin protein, PhaP1, and with soluble PHB (Mw = 350 kDa) in a “high-molecular weight” (HMW) complex and in monomeric/dimeric (M/D) forms with no associated PhaP1 or PHB. Assays for monitoring the formation of PHB in the HMW complex showed no lag phase in CoA release, in contrast to M/D forms of PhaCRe (and PhaCEc), suggesting that PhaC in the HMW fraction has been isolated in a PHB-primed form. The presence of primed and nonprimed PhaC suggests that the elongation rate for PHB formation is also faster than the initiation rate in vivo. A modified micelle model for granule genesis is proposed to accommodate the reported observations.

Class Ib ribonucleotide reductases (RNRs) use a dimanganese-tyrosyl radical cofactor, MnIII2-Y•, in their homodimeric NrdF (β2) subunit to initiate reduction of ribonucleotides to deoxyribonucleotides. The structure of the MnII2 form of NrdF is an important component in understanding O2-mediated formation of the active metallocofactor, a subject of much interest because a unique flavodoxin, NrdI, is required for cofactor assembly. Biochemical studies and sequence alignments suggest that NrdF and NrdI proteins diverge into three phylogenetically distinct groups. The only crystal structure to date of a NrdF with a fully ordered and occupied dimanganese site is that of Escherichia coli MnII2-NrdF, prototypical of the enzymes from actinobacteria and proteobacteria. Here we report the 1.9 Å resolution crystal structure of Bacillus subtilis MnII2-NrdF, representative of the enzymes from a second group, from Bacillus and Staphylococcus. The structures of the metal clusters in the β2 dimer are distinct from those observed in E. coli MnII2-NrdF. These differences illustrate the key role that solvent molecules and protein residues in the second coordination sphere of the MnII2 cluster play in determining conformations of carboxylate residues at the metal sites and demonstrate that diverse coordination geometries are capable of serving as starting points for MnIII2-Y• cofactor assembly in class Ib RNRs.

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.

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 (FnYs, n = 1–4) are promising tools for studying the mechanism of Y· formation and reactivity, as their pKa 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-F3Y) and demonstrate their ability to charge an orthogonal tRNA with a series of FnYs while maintaining high specificity over Y. An evolved aaRS is then used to incorporate FnYs 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 FnY(s), and the resulting proteins are isolated in good yields. FnYs incorporated at position 122 of β2, the site of a stable Y· in wild-type RNR, generate long-lived FnY·s that are characterized by electron paramagnetic resonance (EPR) spectroscopy. Furthermore, we demonstrate that the radical pathway in the mutant Y122(2,3,5)F3Y-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-F3Y· facilitate spectral subtractions that make detection and identification of new Y·s straightforward.

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.

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 (C439) in α2 by a stable diferric tyrosyl radical (Y122•) 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: Y122•→ W48→ Y356 in β2 to Y731→ Y730→ C439 in α2. To study the details of this process, 3-aminotyrosine (NH2Y) has been site-specifically incorporated in place of Y356 of β. The resulting protein, Y356NH2Y-β2, and the previously generated proteins Y731NH2Y-α2 and Y730NH2Y-α2 (NH2Y-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 NH2Y-RNRs and not the result of copurifying wt enzyme. The kinetics of formation of 3-aminotyrosyl radical (NH2Y•) at position 356, 731, and 730 have been measured with all S/E pairs. In all cases, NH2Y• formation is biphasic (kfast of 9–46 s–1 and kslow 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 NH2Y• formation, while the kcat of ∼0.5 s–1 is proposed to be associated with rate-limiting oxidation by NH2Y• of the subsequent amino acid on the pathway during forward PCET. The X-ray crystal structures of Y730NH2Y-α2 and Y731NH2Y-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.

Ribonucleotide reductases (RNRs) convert nucleotides to deoxynucleotides in all organisms. Activity of the class Ia and Ib RNRs requires a stable tyrosyl radical (Y), which can be generated by the reaction of O2 with a diferrous cluster on the β subunit to form active diferric-Y cofactor. Recent experiments have demonstrated, however, that in vivo the class Ib RNR contains an active dimanganese(III)-Y cofactor. The similar metal binding sites of the class Ia and Ib RNRs, their ability to bind both MnII and FeII, and the activity of the class Ib RNR with both diferric-Y and dimanganese(III)-Y cofactors raise the intriguing question of how the cell prevents mismetallation of these essential enzymes. The presence of the class Ib RNR in numerous pathogenic bacteria also highlights the importance of manganese for these organisms’ growth and virulence.

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 (Y122•) cofactor that initiates transfer of the radical to the active site cysteine in α2 (C439), 35 Å removed, is in β2. The oxidation is proposed to involve a hopping mechanism through aromatic amino acids (Y122 → W48 → Y356 in β2 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 β2 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)-α2, β2, CDP, and ATP resulted in detection of NH2Y radical (NH2Y•) intermediates capable of dNDP formation. In this study, the reaction of F3Y356-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 F3Y356-β2, CDP, and ATP has also been examined with NH2Y731-α2 (or NH2Y730-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.

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 (FeIII2-Y•) and dimanganese(III)-Y• (MnIII2-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 MnIII2-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 MnIII2-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.

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.

Human ribonucleotide reductases (hRNRs) catalyze the conversion of nucleotides to deoxynucleotides and are composed of α- and β-subunits that form active αnβm (n, m = 2 or 6) complexes. α 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 (; t1/2 = 23 min). CDP protects α 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 C site binding. Size exclusion chromatography of hRNR or α alone with ClFDP or ClFTP, ± ATP or dGTP, reveals in each case that α forms a kinetically stable hexameric state. This is the first example of hexamerization of α induced by an NDP analog that reversibly binds at the active site.

Cyanophages infecting the marine cyanobacteria Prochlorococcus and Synechococcus encode and express genes for the photosynthetic light reactions. Sequenced cyanophage genomes lack Calvin cycle genes, however, suggesting that photosynthetic energy harvested via phage proteins is not used for carbon fixation. We report here that cyanophages carry and express a Calvin cycle inhibitor, CP12, whose host homologue directs carbon flux from the Calvin cycle to the pentose phosphate pathway (PPP). Phage CP12 was coexpressed with phage genes involved in the light reactions, deoxynucleotide biosynthesis, and the PPP, including a transaldolase gene that is the most prevalent PPP gene in cyanophages. Phage transaldolase was purified to homogeneity from several strains and shown to be functional in vitro, suggesting that it might facilitate increased flux through this key reaction in the host PPP, augmenting production of NADPH and ribose 5-phosphate. Kinetic measurements of phage and host transaldolases revealed that the phage enzymes have kcat/Km values only approximately one third of the corresponding host enzymes. The lower efficiency of phage transaldolase may be a tradeoff for other selective advantages such as reduced gene size: we show that more than half of host-like cyanophage genes are significantly shorter than their host homologues. Consistent with decreased Calvin cycle activity and increased PPP and light reaction activity under infection, the host NADPH/NADP ratio increased two-fold in infected cells. We propose that phage-augmented NADPH production fuels deoxynucleotide biosynthesis for phage replication, and that the selection pressures molding phage genomes involve fitness advantages conferred through mobilization of host energy stores.

Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides and requires a diferric-tyrosyl radical (Y•) cofactor to initiate catalysis. The initiation process requires 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). The rate-limiting step in nucleotide reduction is the conformational gating of the PCET process, which masks the chemistry of radical propagation. 3-Nitrotyrosine (NO2Y) has recently been incorporated site-specifically in place of Y122 in β2. The protein as isolated contained a diferric cluster but no nitrotyrosyl radical (NO2Y•) and was inactive. In the present paper we show that incubation of apo-Y122NO2Y-β2 with Fe2+ and O2 generates a diferric-NO2Y• that has a half-life of 40 s at 25 °C. Sequential mixing experiments, in which the cofactor is assembled to 1.2 NO2Y•/β2 and then mixed with α2, CDP, and ATP, have been analyzed by stopped-flow absorption spectroscopy, rapid freeze quench EPR spectroscopy, and rapid chemical quench methods. These studies have, for the first time, unmasked the conformational gating. They reveal that the NO2Y• is reduced to the nitrotyrosinate with biphasic kinetics (283 and 67 s−1), that dCDP is produced at 107 s−1, and that a new Y• is produced at 97 s−1. Studies with pathway mutants suggest that the new Y• is predominantly located at 356 in β2. In consideration of these data and the crystal structure of Y122NO2Y-β2, a mechanism for PCET uncoupling in NO2Y•-RNR is proposed.

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 Y730 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 NO2Y 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.

Ribonucleotide reductase (RNR, 76 kDa) from Lactobacillus leichmannii is a class II RNR that requires adenosylcobalamin (AdoCbl) as a cofactor. It catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is 100% inactivated by 1 equiv of 2′,2′-difluoro-2′-deoxycytidine 5′-triphosphate (F2CTP) in <2 min. Sephadex G-50 chromatography of the inactivation reaction mixture for 2 min 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, DOI: 10.1021/bi902132u]. After 1 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 [1′-3H]F2CTP in the latter pathway, the reaction mixture at 2 min was reduced with NaBH4 (NaB2H4) and the protein separated from the small molecules using a centrifugation device. The small molecules were dephosphorylated and 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 oxygen. High-field ENDOR studies with [1′-2H]F2CTP from the reaction quenched at 30 s revealed a radical that is nucleotide-based. The relationship between this radical and the trapped cytidine analogue provides insight into the nonalkylative pathway for RNR inactivation relative to the alkylative pathway.

Escherichia coli class Ib ribonucleotide reductase (RNR) converts nucleoside 5′-diphosphates to deoxynucleoside 5′-diphosphates and is expressed under iron-limited and oxidative stress conditions. This RNR is composed of two homodimeric subunits: α2 (NrdE), where nucleotide reduction occurs, and β2 (NrdF), which contains an unidentified metallocofactor that initiates nucleotide reduction. nrdE and nrdF are found in an operon with nrdI, which encodes an unusual flavodoxin proposed to be involved in metallocofactor biosynthesis and/or maintenance. Ni affinity chromatography of a mixture of E. coli (His)6-NrdI and NrdF demonstrated tight association between these proteins. To explore the function of NrdI and identify the metallocofactor, apoNrdF was loaded with MnII and incubated with fully reduced NrdI (NrdIhq) and O2. Active RNR was rapidly produced with 0.25 ± 0.03 tyrosyl radical (Y·) per β2 and a specific activity of 600 units/mg. EPR and biochemical studies of the reconstituted cofactor suggest it is MnIII2-Y·, which we propose is generated by MnII2-NrdF reacting with two equivalents of HO2−, produced by reduction of O2 by NrdF-bound NrdIhq. In the absence of NrdIhq, with a variety of oxidants, no active RNR was generated. By contrast, a similar experiment with apoNrdF loaded with FeII and incubated with O2 in the presence or absence of NrdIhq gave 0.2 and 0.7 Y·/β2 with specific activities of 80 and 300 units/mg, respectively. Thus NrdIhq hinders FeIII2-Y· cofactor assembly in vitro. We propose that NrdI is an essential player in E. coli class Ib RNR cluster assembly and that the MnIII2-Y· cofactor, not the diferric-Y· one, is the active metallocofactor in vivo.

Ribonucleotide reductase (RNR) from Lactobacillus leichmannii, a 76 kDa monomer using adenosylcobalamin (AdoCbl) as a cofactor, catalyzes the conversion of nucleoside triphosphates to deoxynucleotides and is rapidly (<30 s) inactivated by 1 equiv of 2′,2′-difluoro-2′-deoxycytidine 5′-triphosphate (F2CTP). [1′-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 RNR and that 0.71 equiv of cytosine 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 mixture of [1′-3H]F2CTP and [1′-2H]F2CTP followed by reduction with NaBH4, alkylation with iodoacetamide, trypsin digestion, and HPLC separation of the resulting peptides allowed isolation and identification by MALDI-TOF mass spectrometry (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 cysteines were cross-linked to a furanone species derived from the sugar of F2CTP. Incubation of [1′-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 studies and the ones in the preceding paper (DOI: 10.1021/bi9021318) allow proposal of a mechanism of inactivation of RNR by F2CTP involving multiple reaction pathways. The proposed mechanisms share many common features with F2CDP inactivation of the class I RNRs.

Escherichia coli ribonucleotide reductase (RNR), an α2β2 complex, catalyzes the conversion of nucleoside 5′-diphosphate substrates (S) to 2′-deoxynucleoside 5′-diphosphates. α2 houses the active site for nucleotide reduction and the binding sites for allosteric effectors (E). β2 contains the essential diferric tyrosyl radical (Y122•) cofactor which, in the presence of S and E, oxidizes C439 in α to a thiyl radical, C439•, to initiate nucleotide reduction. This oxidation occurs over 35 Å and is proposed to involve a specific pathway: Y122• → W48 → Y356 in β2 to Y731 → Y730 → C439 in α2. 3-Aminotyrosine (NH2Y) has been site-specifically incorporated at residues 730 and 731, and formation of the aminotyrosyl radical (NH2Y•) has been examined by stopped-flow (SF) UV−vis and EPR spectroscopies. To examine the pathway dependence of radical propagation, the double mutant complexes Y356F-β2:Y731NH2Y-α2, Y356F-β2:Y730NH2Y-α2, and wt-β2:Y731F/Y730NH2Y-α2, in which the nonoxidizable F acts as a pathway block, were studied by SF and EPR spectroscopies. In all cases, no NH2Y• was detected. To study off-pathway oxidation, Y413, located 5 Å from Y730 and Y731 but not implicated in long-range oxidation, was examined. Evidence for NH2Y413• was sought in three complexes: wt-β2:Y413NH2Y-α2 (a), wt-β2:Y731F/Y413NH2Y-α2 (b), and Y356F-β2:Y413NH2Y-α2 (c). With (a), NH2Y• 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 NH2Y• formation was 0.2−0.3% of that observed at 731 and 730, and with (c), no NH2Y• was observed. These studies suggest the evolution of an optimized pathway of conserved Ys in the oxidation of C439.

Polyhydroxybutyrate (PHB) synthases catalyze the conversion of 3-hydroxybutyryl coenzyme A (HBCoA) to PHB with a molecular mass of 1.5 MDa. The class III synthase from Allochromatium vinosum is a tetramer of PhaEPhaC (each 40 kDa). The polymerization involves covalent catalysis using C149 of PhaC with one PHB chain per PhaEC dimer. Two mechanisms for elongation have been proposed. The first involves an active site composed of two monomers in which the growing hydroxybutyrate (HB) chain alternates between C149 on each monomer. The second involves C149 and covalent and noncovalent (HB)nCoA intermediates. Two approaches were investigated to distinguish between these models. The first involved the wild-type (wt) PhaEC primed with sTCoA [a CoA ester of (HB)3 in which the terminal HO group is replaced with an H] which uniformly loads the enzyme. The primed synthase was reacted with [1-14C]HBCoA by a rapid chemical quench method and analyzed for covalent and noncovalent intermediates. Radiolabel was found only with the protein. The second approach used C149S-PhaEC which catalyzes polymer formation at 1/2200 of the rate of wt-PhaEC (1.79 min−1 vs 3900 min−1). C149S-PhaEC was incubated with [1-14C]HBCoA and chemically quenched on the minute time scale to reveal noncovalently bound [1-14C](HB)2CoA and (HB)3CoA as well as covalently labeled protein. Synthesized (HB)nCoA (n = 2 or 3) was shown to acylate PhaEC with rate constants of 1−2 min−1, and these species were converted into polymer. Thus, the (HB)nCoA analogues function as kinetically and chemically competent intermediates. These results support the mechanism involving covalently and noncovalently bound intermediates.

Gemcitabine 5′-diphosphate (F2CDP) is a potent inhibitor of ribonucleotide reductases (RNRs), enzymes that convert nucleotides (NDPs) to deoxynucleotides and are essential for DNA replication and repair. The Escherichia coli RNR, an α2β2 complex, when incubated with 1 equiv of F2CDP catalyzes the release of two fluorides and cytosine concomitant with enzyme inactivation. In the presence of reductant (thioredoxin/thioredoxin reductase/NADPH or DTT), the enzyme inactivation results from its covalent labeling of α with the sugar of F2CDP (one label/α2β2). SDS-PAGE analysis of the inactivated RNR without boiling of the sample reveals that α migrates as an 87 and 110 kDa protein in a ratio of 0.6:0.4. When the reductant is omitted, RNR is inactivated by loss of the essential tyrosyl radical and formation of a new radical. Inactivation studies with C225S-α in the presence or absence of reductants, reveal it behaves like wt-RNR in the absence of reductant. Inactivated C225S-α migrates as an 87 kDa protein and is not covalently modified. C225 is one of the cysteines in RNR’s active site that supplies reducing equivalents to make dNDPs. To identify the new radical formed, [1′-2H]-F2CDP was studied with wt- and C225S-RNR by 9 and 140 GHz EPR spectroscopy. These studies revealed that the new radical is a nucleotide derived with g values of gx 2.00738, gy 2.00592, and gz 2.00230 and with altered hyperfine interactions (apparent triplet collapsed to a doublet) relative to [1′-1H]-F2CDP. The EPR features are very similar to those we recently reported for the nucleotide radical generated with CDP and E441Q-RNR.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleoside 5′-diphosphates to the corresponding deoxynucleotides supplying the dNTPs required for DNA replication and DNA repair. Class I RNRs require two subunits, α and β, for activity. Humans possess two β subunits: one involved in S phase DNA replication (β) and a second in mitochondrial DNA replication (β′ or p53R2) and potentially DNA repair. Gemcitabine (F2C) is used clinically as an anticancer agent, and its phosphorylated metabolites target many enzymes involved in nucleotide metabolism, including RNR. The present investigation with α (specific activity of 400 nmol min−1 mg−1) and β′ (0.6 Y·/β′2 and a specific activity of 420 nmol min−1 mg−1) establishes that F2CDP is a substoichiometric inactivator of RNR. Incubation of this α/β′ with [1′-3H]-F2CDP or [5-3H]-F2CDP and reisolation of the protein by Sephadex G-50 chromatography resulted in recovery 0.5 equiv of covalently bound sugar and 0.03 equiv of tightly associated cytosine to α2. SDS−PAGE analysis (loaded without boiling) of the inactivated RNR showed that 60% of α migrates as a 90 kDa protein and 40% as a 120 kDa protein. Incubation of [1′-3H]-F2CDP with active site mutants C444S/A, C218S/A, and E431Q/D-α and the C-terminal tail C787S/A and C790S/A mutants reveals that no sugar label is bound to the active site mutants of α and that, in the case of C218S-α, α migrates as a 90 kDa protein. Analysis of the inactivated wt-α/β′ RNR by size exclusion chromatography indicates a quaternary structure of α6β′6. A mechanism of inactivation common with hα/β is presented.

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 α2 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 × 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.

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, α2 and β2. The interactions between α2 and β2 are thought to be largely associated with the C-terminal 20 amino acids (residues 356−375) of β2. To study subunit interactions, a single reactive cysteine has been introduced into each of 15 positions along the C-terminal tail of β2. 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-β2 has been incubated with 1 equiv of α2 and photolyzed, and the results have been analyzed quantitatively by SDS−PAGE. Each DAN-β2 was incubated with a 50-fold excess of α2, 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-β2 variants (351, 356, 365, and 367) with α2 gave a Kd ≈ 0.4 μM for subunit interaction. Disruption of the interaction of the α2–DAN-β2 complex is accompanied by a decrease in fluorescence intensity and can serve as a high-throughput screen for inhibitors of subunit interactions.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. The Escherichia coli class Ia RNR is composed of α and β subunits that form an α2β2 active complex. β contains the diferric tyrosyl radical (Y•) cofactor that is essential for the reduction process that occurs on α. [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 β 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, [β] could be varied from 4 to 3300 µM. [Y•] in vivo and on affinity-purified Strep-β in vitro was determined by EPR spectroscopy and Western analysis. In both cases, there was 0.1–0.3 Y• radical per β. To determine if the substoichiometric Y• level was associated with apo β or diferric β, 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 O2 to assemble the cofactor and EPR analysis to quantitate Y•, revealed that β is completely loaded with a diferric cluster even when its concentration in vivo is 244 µM. These titrations, furthermore, resulted in 1 Y• radical per β, the highest levels reported. Whole cell Mössbauer analysis on cells induced with 0.5 mM arabinose supports high iron loading in β. 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 conversion of nucleoside 5′-diphosphates to deoxynucleoside 5′-diphosphates and is a 1:1 complex of two homodimeric subunits: α2 and β2. As a first step towards mapping the subunit interface, β2 (V365C) was labeled with [14C]-benzophenone (BP) iodoacetamide. The resulting [14C]-BP–β2 (V365C) was complexed with α2 and irradiated at 365 nm for 30 min at 4 °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 [14C]-labeled peptides were identified: two contained a peptide in β to which the BP was attached. The third contained the same β peptide and a peptide in α found in its αD helix. These results provide direct support for the proposed docking model of α2β2.Molecular insight into the subunit interface of Escherichia coli RNR using peptide mapping is presented.

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 Ib 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 Ib RNR. It possesses redox properties unprecedented for a flavodoxin (Eox/sq = −264 ± 17 mV and Esq/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.

Structures of active RNR, a 1:1 complex of R1 and R2, are not available. However, Uhlin and Eklund6 have generated a docking model of this complex from the individual structures of R1 and R2 (refs. 7,8). Their analysis, along with Clustal W alignments of over 160 class I RNR sequences, have suggested a pathway for reversible radical migration, which involves the following residues and perhaps the diiron center: [Y122 ⇆ Fe cluster ⇆ W48 ⇆ Y356] in R2 and [Y731 ⇆ Y730 ⇆ C439 ⇆ substrate] in R1 (Fig. 1) (refs. 5,6). Site-directed mutagenesis has been employed to provide evidence that these residues are essential for radical initiation9, 10, 11, 12, 13. However, these mutants have not been mechanistically informative, as they exhibit only marginal catalytic activity probably associated to residual wild-type contamination of the purified mutants. Of the 19 natural amino acids that can be substituted for Tyr at this position, only Trp and Cys have oxidation potentials close to that of Tyr, but they are poor structural substitutes5. To generate mechanistically more meaningful mutations, we aimed to replace residue Y356 on the proposed radical initiation pathway in R2 with unnatural amino acids that sterically resemble Tyr and have varying oxidation potentials and pK as of their phenolic side chains.To this end, we explored the properties of FnYs (n = 1–4) and showed that they may serve as probes for disentangling the reactivities of redox-active Tyr residues in proteins (Fig. 2) (see ref. 14). FnYs are isosteric with tyrosine, present a range of phenolic pK as as well as a large range of oxidation potentials (Table 1). With the proper choice of FnY and reaction pH, one can change the protonation state of FnY356, its oxidation potential or both. Thus, FnYs are excellent probes for examining proton-coupled electron transfer mechanisms in radical propagation and should be generally useful for studying proteins that require stable or transient tyrosyl radicals for catalysis15, 16. We next developed an intein-mediated semisynthesis of R2 that allows site-specific replacement of residue Y356 in R2 with FnYs17, 18.To demonstrate how EPL works, first the mechanism of intein self-expulsion must be outlined. The proposed mechanism for the S. cerevisiae Vma intein is shown in Figure 3 (see refs. 24,25). Splicing is initiated by an N-S acyl shift that converts a peptide bond into a thioester at the N-extein junction site. Transthioesterification via the C-extein Cys residue results in a branched intermediate. This intermediate undergoes excision of the intein domain by attack of an Asn residue on the peptide bond at the C-extein junction site forming an intein succinimide and the spliced extein domains. Spontaneous S-N acyl rearrangement generates the ligated N- and C-exteins connected via an amide bond.EPL also has a number of limitations. First, site-specific incorporation of probes can only occur close to the N or C termini of the protein because peptides longer than 40–50 residues are difficult to synthesize and purify. Second, the site of ligation must be accessible to solvent in the native protein. If the site of ligation is buried, then the ligation reaction needs to be carried out under denaturing conditions, and methods to refold the protein are required. Third, the thioester-truncated protein undergoes hydrolysis in competition with ligation to the peptide. The rate of ligation, a bimolecular reaction, is elevated by increasing concentrations. However, the ligation is still slow and consequently hydrolysis almost always accompanies the desired reaction. Methods are thus required to separate the truncated protein from the full-length, ligated protein. This problem is aggravated if the protein of interest is multimeric, as in the case of R2.1. Synthesis and purification of FnY(s). This block is covered in Steps 1–9 of PROCEDURE.2. Protection of the amino group of FnY(s) with the 9-fluorenylmethoxy carbonyl (Fmoc)-protecting group and synthesis and purification of the peptide-22mer (H2N–CS(FnY)LVGQIDSEVDTDDLSNFQL-COOH) containing desired FnY(s), Steps 10–34.3. Growth of MESNA-activated, truncated R2 and ligation to the FnY-22mer(s), Steps 35–50.The timing for incorporation of a single FnY into R2 is indicated below. Note that synthesis of F4Y requires 4 weeks.Reagent and equipment setup (including expression and purification of TPL): 1 monthSynthesis and purification of an FnY (other than F4Y) in Figure 2: 1 weekSynthesis and purification of Fmoc-FnY and FnY-22mer: 3 weeksSemisynthesis and purification of FnY-R2: 3–4 weeksTroubleshooting advice can be found in Table 3.A typical 1 liter enzymatic synthesis of 3,5-F2Y yields 1.7 g of the desired product (80% yield). Addition of the Fmoc-protecting group by the described procedure yields Fmoc-3,5-F2Y-OH in good yield (85%). The 3,5-F2Y-22mer is synthesized by a combination of automatic and manual SPPS. After purification and deprotection of the tButhio-protecting group of the N-terminal Cys, 4.5 μmol of 3,5-F2Y-22mer is obtained from a theoretical yield of 6 μmol (75% yield). Ligation of 4.5 μmol of peptide to 45 mg (0.52 μmol) of MESNA-activated, truncated R2 and subsequent purification yields 20 mg (0.23 μmol) of full-length homodimeric 3,5-F2Y-R2. SDS-PAGE and ESI-MS analyses confirm incorporation of 3,5-F2Y.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. The class I RNRs are composed of two subunits, α and β, with proposed quaternary structures of α2β2, α6β2, or α6β6, depending on the organism. The α subunits bind the nucleoside diphosphate substrates and the dNTP/ATP allosteric effectors that govern specificity and turnover. The β2 subunit houses the diferric Y^• (1 radical per β2) cofactor that is required to initiate nucleotide reduction. 2′,2′-Difluoro-2′-deoxycytidine (F₂C) is presently used clinically in a variety of cancer treatments and the 5′-diphosphorylated F₂C (F₂CDP) is a potent inhibitor of RNRs. The studies with [1′-³H]-F₂CDP and [5-³H]-F₂CDP have established that F₂CDP is a substoichiometric mechanism based inhibitor (0.5 eq F₂CDP/α) of both the Escherichia coli and the human RNRs in the presence of reductant. Inactivation is caused by covalent labeling of RNR by the sugar of F₂CDP (0.5 eq/α) and is accompanied by release of 0.5 eq cytosine/α. Inactivation also results in loss of 40% of β2 activity. Studies using size exclusion chromatography reveal that in the E. coli RNR, an α2β2 tight complex is generated subsequent to enzyme inactivation by F₂CDP, whereas in the human RNR, an α6β6 tight complex is generated. Isolation of these complexes establishes that the weak interactions of the subunits in the absence of nucleotides are substantially increased in the presence of F₂CDP and ATP. This information and the proposed asymmetry between the interactions of αnβn provide an explanation for complete inactivation of RNR with substoichiometric amounts of F₂CDP.

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides and is an essential enzyme for DNA replication and repair. Cells have evolved intricate mechanisms to regulate RNR activity to ensure high fidelity of DNA replication during normal cell-cycle progression and of DNA repair upon genotoxic stress. The RNR holoenzyme is composed of a large subunit R1 (α, oligomeric state unknown) and a small subunit R2 (β2). R1 binds substrates and allosteric effectors; R2 contains a diferric-tyrosyl radical [(Fe)2-Y·] cofactor that is required for catalysis. In Saccharomyces cerevisiae, R1 is predominantly localized in the cytoplasm, whereas R2, which is a heterodimer (ββ′), is predominantly in the nucleus. When cells encounter DNA damage or stress during replication, ββ′ is redistributed from the nucleus to the cytoplasm in a checkpoint-dependent manner, resulting in the colocalization of R1 and R2. We have identified two proteins that have an important role in ββ′ nuclear localization: the importin β homolog Kap122 and the WD40 repeat protein Wtm1. Deletion of either WTM1 or KAP122 leads to loss of ββ′ nuclear localization. Wtm1 and its paralog Wtm2 are both nuclear proteins that are in the same protein complex with ββ′. Wtm1 also interacts with Kap122 in vivo and requires Kap122 for its nuclear localization. Our results suggest that Wtm1 acts either as an adaptor to facilitate nuclear import of ββ′ by Kap122 or as an anchor to retain ββ′ in the nucleus.

Bleomycins are a family of glycopeptide antibiotics that have potent antitumour activity against a range of lymphomas, head and neck cancers and germ-cell tumours. The therapeutic efficacy of the bleomycins is limited by development of lung fibrosis. The cytotoxic and mutagenic effects of the bleomycins are thought to be related to their ability to mediate both single-stranded and double-stranded DNA damage, which requires the presence of specific cofactors (a transition metal, oxygen and a one-electron reductant). Progress in understanding the mechanisms involved in the therapeutic efficacy of the bleomycins and the unwanted toxicity and elucidation of the biosynthetic pathway of the bleomycins sets the stage for developing a more potent, less toxic therapeutic agent.

The bleomycins (BLMs) are used clinically in combination chemotherapy, their clinical usefulness being limited by the accompanying pulmonary toxicity. Much has been learned about the structure and function of BLMs in vitro. However, the mechanism of their cytoxicity in vivo, including their target(s), metal cofactor(s) effecting nucleic acid cleavage and its (their) oxidation state, concentrations of BLM in the nucleus of the cell, BLM metabolism, hot spots for double-strand DNA cleavage, and their repair, have remained elusive. New methods offer new opportunities to revisit and solve old problems, which could ultimately lead to development of a more effective therapeutic.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms, providing the monomeric precursors required for DNA replication and repair. The class I RNRs are composed of two subunits; the R1 subunit contains the active site for nucleotide reduction and allosteric effector binding sites, whereas the R2 subunit houses the essential diirontyrosyl (Y·) radical cofactor. A major unresolved issue is the mechanism by which the tyrosyl radical on R2 (Y122, Escherichia coli numbering) reversibly generates the transient thiyl radical (S·) on R1 that initiates nucleotide reduction. This intersubunit radical initiation is postulated to occur through a defined pathway involving conserved aromatic amino acids (R2: Y122, W48, Y356; R1: Y731, Y730) over a long distance of 35 Å. A 20-mer peptide identical to the C-terminal tail of R2 (356–375) and containing Y356 is a competitive inhibitor with respect to R2, and it effectively blocks nucleotide reduction. We now report that a 21-mer peptide, in which a tryptophan has been incorporated at the N terminus of the 20th mer, can replace the R2 subunit and initiate nucleotide reduction by photoinitiated radical generation. The deoxynucleotide generated depends on the presence of allosteric effector and is pathway-dependent. Replacement of Y731 of R2 with phenylalanine prevents deoxynucleotide formation. These results provide direct evidence for the chemical competence of aromatic amino acid radicals and the importance of Y356 in R2 in the radical initiation process of the class I RNRs.

Covering: 1989 to 2003

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides. Class I RNRs are composed of two homodimeric subunits: R1 and R2. R1 is directly involved in the reduction, and R2 contains the diferric-tyrosyl radical (Y⋅) cofactor essential for the initiation of reduction. Saccharomyces cerevisiae has two RNRs; Y1 and Y3 correspond to R1, whereas Y2 and Y4 correspond to R2. Y4 is essential for diferric-Y⋅ formation in Y2 from apoY2, Fe2+, and O2. The actual function of Y4 is controversial. Y2 and Y4 have been further characterized in an effort to understand their respective roles in nucleotide reduction. (His)6-Y2, Y4, and (His)6-Y4 are homodimers, isolated largely in apo form. Their CD spectra reveal that they are predominantly helical. The concentrations of Y2 and Y4 in vivo are 0.5–2.3 μM, as determined by Western analysis. Incubation of Y2 and Y4 under physiological conditions generates apo Y2Y4 heterodimer, which can form a diferric-Y⋅ when incubated with Fe2+ and O2. Holo Y2Y4 heterodimer contains 0.6–0.8 Y⋅ and has a specific activity of 0.8–1.3 μmol⋅min⋅mg. Titration of Y2 with Y4 in the presence of Fe2+ and O2 gives maximal activity with one equivalent of Y4 per Y2. Models for the function of Y4 based on these data and the accompanying structure will be discussed.

Structural studies, sequence alignments, and biochemistry have provided new insights into the evolution of the purine biosynthetic pathway. The importance of chemistry, the binding of ribose 5-phosphate (common to all purine biosynthetic intermediates), and transient protein–protein interactions in channeling of chemically unstable intermediates have all been examined in the past few years.

Ribonucleotide reductases (RNRs) utilize radical chemistry to reduce nucleotides to deoxynucleotides in all organisms. In the class Ia and Ib RNRs, this reaction requires a stable tyrosyl radical (Y•) generated by oxidation of a reduced dinuclear metal cluster. The FeIII2-Y• cofactor in the NrdB subunit of the class Ia RNRs can be generated by self-assembly from FeII2-NrdB, O2, and a reducing equivalent. By contrast, the structurally homologous class Ib enzymes require a MnIII2-Y• cofactor in their NrdF subunit. MnII2-NrdF does not react with O2, but it binds the reduced form of a conserved flavodoxin-like protein, NrdIhq, which, in the presence of O2, reacts to form the MnIII2-Y• cofactor. Here we investigate the mechanism of assembly of the MnIII2-Y• cofactor in Bacillus subtilis NrdF. Cluster assembly from MnII2-NrdF, NrdIhq, and O2 has been studied by stopped flow absorption and rapid freeze quench EPR spectroscopies. The results support a mechanism in which NrdIhq reduces O2 to O2•– (40–48 s–1, 0.6 mM O2), the O2•– channels to and reacts with MnII2-NrdF to form a MnIIIMnIV intermediate (2.2 ± 0.4 s–1), and the MnIIIMnIV species oxidizes tyrosine to Y• (0.08–0.15 s–1). Controlled production of O2•– by NrdIhq during class Ib RNR cofactor assembly both circumvents the unreactivity of the MnII2 cluster with O2 and satisfies the requirement for an “extra” reducing equivalent in Y• generation.

Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides to provide the monomeric building blocks for DNA replication and repair. Nucleotide reduction occurs by way of multistep proton-coupled electron transfer (PCET) over a pathway of redox active amino acids spanning ∼35 Å and two subunits (α2 and β2). Despite the fact that PCET in RNR is rapid, slow conformational changes mask examination of the kinetics of these steps. As such, we have pioneered methodology in which site-specific incorporation of a [ReI] photooxidant on the surface of the β2 subunit (photoβ2) allows photochemical oxidation of the adjacent PCET pathway residue β-Y356 and time-resolved spectroscopic observation of the ensuing reactivity. A series of photoβ2s capable of performing photoinitiated substrate turnover have been prepared in which four different fluorotyrosines (FnYs) are incorporated in place of β-Y356. The FnYs are deprotonated under biological conditions, undergo oxidation by electron transfer (ET), and provide a means by which to vary the ET driving force (ΔG°) with minimal additional perturbations across the series. We have used these features to map the correlation between ΔG° and kET both with and without the fully assembled photoRNR complex. The photooxidation of FnY356 within the α/β subunit interface occurs within the Marcus inverted region with a reorganization energy of λ ≈ 1 eV. We also observe enhanced electronic coupling between donor and acceptor (HDA) in the presence of an intact PCET pathway. Additionally, we have investigated the dynamics of proton transfer (PT) by a variety of methods including dependencies on solvent isotopic composition, buffer concentration, and pH. We present evidence for the role of α2 in facilitating PT during β-Y356 photooxidation; PT occurs by way of readily exchangeable positions and within a relatively “tight” subunit interface. These findings show that RNR controls ET by lowering λ, raising HDA, and directing PT both within and between individual polypeptide subunits.

Proton-coupled electron transfer (PCET) is a fundamental mechanism important in a wide range of biological processes including the universal reaction catalysed by ribonucleotide reductases (RNRs) in making de novo, the building blocks required for DNA replication and repair. These enzymes catalyse the conversion of nucleoside diphosphates (NDPs) to deoxynucleoside diphosphates (dNDPs). In the class Ia RNRs, NDP reduction involves a tyrosyl radical mediated oxidation occurring over 35 Å across the interface of the two required subunits (β2 and α2) involving multiple PCET steps and the conserved tyrosine triad [Y356(β2)–Y731(α2)–Y730(α2)]. We report the synthesis of an active photochemical RNR (photoRNR) complex in which a Re(I)-tricarbonyl phenanthroline ([Re]) photooxidant is attached site-specifically to the Cys in the Y356C-(β2) subunit and an ionizable, 2,3,5-trifluorotyrosine (2,3,5-F3Y) is incorporated in place of Y731 in α2. This intersubunit PCET pathway is investigated by ns laser spectroscopy on [Re356]-β2:2,3,5-F3Y731-α2 in the presence of substrate, CDP, and effector, ATP. This experiment has allowed analysis of the photoinjection of a radical into α2 from β2 in the absence of the interfacial Y356 residue. The system is competent for light-dependent substrate turnover. Time-resolved emission experiments reveal an intimate dependence of the rate of radical injection on the protonation state at position Y731(α2), which in turn highlights the importance of a well-coordinated proton exit channel involving the key residues, Y356 and Y731, at the subunit interface.