Class Ia Ribonucleotide Reductase Research Articles

Structural insights into the initiation of free radical formation in the Class Ib ribonucleotide reductases in Mycobacteria

Class I ribonucleotide reductases consisting of α and β subunits convert ribonucleoside diphosphates to deoxyribonucleoside diphosphates involving an intricate free radical mechanism. The generation of free radicals in the Class Ib ribonucleotide reductases is mediated by di-manganese ions in the β subunits and is externally assisted by flavodoxin-like NrdI subunit. This is unlike Class Ia ribonucleotide reductases, where the free radical generation is initiated at its di-iron centre in the β subunits with no external support from another subunit. Class 1b ribonucleotide reductase complex is an essential enzyme complex in the human pathogen Mycobacterium tuberculosis and its structural details are largely unknown. In this study we have determined the crystal structures of Mycobacterial NrdI in oxidized and reduced forms, and similarly those of NrdF2:NrdI complexes. These structures provide detailed atomic view of the mechanism of free radical generation in the β subunit in this pathogen. We observe a well-formed channel in NrdI from the surface leading to the buried FMN moiety and propose that oxygen molecule accesses FMN through it. The oxygen molecule is further converted to a superoxide ion upon electron transfer at the FMN moiety. Similarly, a path for superoxide radical transfer between NrdI and NrdF2 is also observed. The oxidation of Mn(II) in NrdF2I to high valent oxidation state (either Mn(III) or Mn(IV) assisted by the reduced FMN site was evidently confirmed by EPR studies. SEC-MALS and low resolution cryo-EM map indicate unusual stoichiometry of 2:1 in the M. tuberculosis NrdF2I complex. A density close to Tyr 110 at a distance <2.3Å is observed, which we interpret as OH group. Overall, the study therefore provides important clues on the initiation of free radical generation in the β subunit of the ribonucleotide reductase complex in M. tuberculosis.

How ATP and dATP Act as Molecular Switches to Regulate Enzymatic Activity in the Prototypical Bacterial Class Ia Ribonucleotide Reductase.

Class Ia ribonucleotide reductases (RNRs) are allosterically regulated by ATP and dATP to maintain the appropriate deoxyribonucleotide levels inside the cell for DNA biosynthesis and repair. RNR activity requires precise positioning of the β2 and α2 subunits for the transfer of a catalytically essential radical species. Excess dATP inhibits RNR through the creation of an α-β interface that restricts the ability of β2 to obtain a position that is capable of radical transfer. ATP breaks the α-β interface, freeing β2 and restoring enzyme activity. Here, we investigate the molecular basis for allosteric activity regulation in the well-studied Escherichia coli class Ia RNR through the determination of six crystal structures and accompanying biochemical and mutagenesis studies. We find that when dATP is bound to the N-terminal regulatory cone domain in α, a helix unwinds, creating a binding surface for β. When ATP displaces dATP, the helix rewinds, dismantling the α-β interface. This reversal of enzyme inhibition requires that two ATP molecules are bound in the cone domain: one in the canonical nucleotide-binding site (site 1) and one in a site (site 2) that is blocked by phenylalanine-87 and tryptophan-28 unless ATP is bound in site 1. When ATP binds to site 1, histidine-59 rearranges, prompting the movement of phenylalanine-87 and trytophan-28, and creating site 2. dATP hydrogen bonds to histidine-59, preventing its movement. The importance of site 2 in the restoration of RNR activity by ATP is confirmed by mutagenesis. These findings have implications for the design of bacterial RNR inhibitors.

Theoretical Insights into the Generation Mechanism of the Tyr122 Radical Catalyzed by Intermediate X in Class Ia Ribonucleotide Reductase.

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides in all organisms. There is an ∼35 Å long-range electron-hole transfer pathway during the catalytic process of class Ia RNR, which can be described as Tyr122β ↔ [Trp48β]? ↔ Tyr356β ↔ Tyr731α ↔ Tyr730α ↔ Cys439α. The formation of the Y122• radical initiates this long-range radical transfer process. However, the generation mechanism of Y122• is not yet clear due to confusion over the intermediate X structures. Based on the two reported X structures, we examined the possible mechanisms of Y122• generation by density functional theory (DFT) calculations. Our examinations revealed that the generation of the Y122• radical from the two different core structures of X was via a similar two-step reaction, with the first step of proton transfer for the formation of the proton receptor of Y122 and the second step of a proton-coupled long-range electron transfer reaction with the proton transfer from the Y122 hydroxyl group to the terminal hydroxide ligand of Fe1III and simultaneously electron transfer from the side chain of Y122 to Fe2IV. These findings provide an insight into the formation mechanism of Y122• catalyzed by the double-iron center of the β subunit of class Ia RNR.

Radical Transport Facilitated by a Proton Transfer Network at the Subunit Interface of Ribonucleotide Reductase.

Ribonucleotide reductases (RNRs) play an essential role in the conversion of nucleotides to deoxynucleotides in all organisms. The Escherichia coli class Ia RNR requires two homodimeric subunits, α and β. The active form is an asymmetric αα'ββ' complex. The α subunit houses the site for nucleotide reduction initiated by a thiyl radical (C439•), and the β subunit houses the diferric-tyrosyl radical (Y122•) that is essential for C439• formation. The reactions require a highly regulated and reversible long-range proton-coupled electron transfer pathway involving Y122•[β] ↔ W48?[β] ↔ Y356[β] ↔ Y731[α] ↔ Y730[α] ↔ C439[α]. In a recent cryo-EM structure, Y356[β] was revealed for the first time and it, along with Y731[α], spans the asymmetric α/β interface. An E52[β] residue, which is essential for Y356 oxidation, allows access to the interface and resides at the head of a polar region comprising R331[α], E326[α], and E326[α'] residues. Mutagenesis studies with canonical and unnatural amino acid substitutions now suggest that these ionizable residues are important in enzyme activity. To gain further insights into the roles of these residues, Y356• was photochemically generated using a photosensitizer covalently attached adjacent to Y356[β]. Mutagenesis studies, transient absorption spectroscopy, and photochemical assays monitoring deoxynucleotide formation collectively indicate that the E52[β], R331[α], E326[α], and E326[α'] network plays the essential role of shuttling protons associated with Y356 oxidation from the interface to bulk solvent.

Structural investigation of 2'-azido-2'-deoxyribonucleoside-5'-diphosphate inhibition of class Ia Escherichia coli ribonucleotide reductase.

Bacterial class Ia ribonucleotide reductases (RNRs) are attractive antibiotic targets due to their essential role in the biosynthesis of deoxyribonucleotides from the corresponding ribonucleotides. The catalytically active state of class Ia RNRs consists of a heterodimer of two homodimers, α;2 and β;2, in which the α;2 subunit contains substrate-binding and allosteric sites and the β;2 subunit contains two di-iron sites responsible for the initial steps of a 32-Å radical transfer pathway that enables conversion of the ribonucleotide substrate. Recent structural work in the Drennan lab has elucidated the active state structure of this enzyme, but required a doubly-mutated variant of β;2, E52Q/F3Y122-β;2, to decrease the rate of subunit dissociation. We describe here the use of 2′-azido-2′-deoxyribonucleoside-5′-diphosphate, a nucleotide analog, to trap the active state structure of the wild type Escherichia coli class Ia RNR and subsequent structural efforts to elucidate the conformational trajectory of the dissociation. This work provides a basis for understanding the complex regulatory mechanisms of RNR, which may result in novel antibiotic targets.

A rapid and sensitive assay for quantifying the activity of both aerobic and anaerobic ribonucleotide reductases acting upon any or all substrates.

Ribonucleotide reductases (RNRs) use radical-based chemistry to catalyze the conversion of all four ribonucleotides to deoxyribonucleotides. The ubiquitous nature of RNRs necessitates multiple RNR classes that differ from each other in terms of the phosphorylation state of the ribonucleotide substrates, oxygen tolerance, and the nature of both the metallocofactor employed and the reducing systems. Although these differences allow RNRs to produce deoxyribonucleotides needed for DNA biosynthesis under a wide range of environmental conditions, they also present a challenge for establishment of a universal activity assay. Additionally, many current RNR assays are limited in that they only follow the conversion of one ribonucleotide substrate at a time, but in the cell, all four ribonucleotides are actively being converted into deoxyribonucleotide products as dictated by the cellular concentrations of allosteric specificity effectors. Here, we present a liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based assay that can determine the activity of both aerobic and anaerobic RNRs on any combination of substrates using any combination of allosteric effectors. We demonstrate that this assay generates activity data similar to past published results with the canonical Escherichia coli aerobic class Ia RNR. We also show that this assay can be used for an anaerobic class III RNR that employs formate as the reductant, i.e. Streptococcus thermophilus RNR. We further show that this class III RNR is allosterically regulated by dATP and ATP. Lastly, we present activity data for the simultaneous reduction of all four ribonucleotide substrates by the E. coli class Ia RNR under various combinations of allosteric specificity effectors. This validated LC-MS/MS assay is higher throughput and more versatile than the historically established radioactive activity and coupled RNR activity assays as well as a number of the published HPLC-based assays. The presented assay will allow for the study of a wide range of RNR enzymes under a wide range of conditions, facilitating the study of previously uncharacterized RNRs.

19F Electron-Nuclear Double Resonance Reveals Interaction between Redox-Active Tyrosines across the α/β Interface of E. coli Ribonucleotide Reductase.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, thereby playing a key role in DNA replication and repair. Escherichia coli class Ia RNR is an α2β2 enzyme complex that uses a reversible multistep radical transfer (RT) over 32 Å across its two subunits, α and β, to initiate, using its metallo-cofactor in β2, nucleotide reduction in α2. Each step is proposed to involve a distinct proton-coupled electron-transfer (PCET) process. An unresolved step is the RT involving Y356(β) and Y731(α) across the α/β interface. Using 2,3,5-F3Y122-β2 with 3,5-F2Y731-α2, GDP (substrate) and TTP (allosteric effector), a Y356• intermediate was trapped and its identity was verified by 263 GHz electron paramagnetic resonance (EPR) and 34 GHz pulse electron–electron double resonance spectroscopies. 94 GHz 19F electron-nuclear double resonance spectroscopy allowed measuring the interspin distances between Y356• and the 19F nuclei of 3,5-F2Y731 in this RNR mutant. Similar experiments with the double mutant E52Q/F3Y122-β2 were carried out for comparison to the recently published cryo-EM structure of a holo RNR complex. For both mutant combinations, the distance measurements reveal two conformations of 3,5-F2Y731. Remarkably, one conformation is consistent with 3,5-F2Y731 within the H-bond distance to Y356•, whereas the second one is consistent with the conformation observed in the cryo-EM structure. The observations unexpectedly suggest the possibility of a colinear PCET, in which electron and proton are transferred from the same donor to the same acceptor between Y356 and Y731. The results highlight the important role of state-of-the-art EPR spectroscopy to decipher this mechanism.

Structural Investigation of Allosteric Regulation in Class III Ribonucleotide Reductases

Ribonucleotide reductases (RNRs) are essential enzymes that use radical‐based chemistry to catalyze the reduction of ribonucleotides to deoxyribonucleotides. Each RNR class uses a different cofactor to generate a catalytically‐essential thiyl radical species in the enzyme active site. Anaerobic (class III) RNRs employ an oxygen‐sensitive glycyl radical cofactor installed within the glycyl radical domain (GRD) that is adjacent to the active site, whereas aerobic (class Ia) RNRs employ long‐range radical transfer between the catalytic subunit and a di‐iron/tyrosyl radical cofactor housed in the b2 subunit. To maintain intracellular nucleotide pool balance, RNRs are allosterically regulated. For the prototypical class Ia RNRs from Escherichia coli, this allosteric activity regulation involves dATP binding to the N‐terminus of the catalytic subunit in a region known as the ‘cone domain’, resulting in an association between the cone domain and the b2 subunit that prevents radical transfer between subunits. Despite not requiring a b2 subunit, some class III RNRs (e.g. Streptococcus thermophilus)have a cone domain and are allosterically inhibited by dATP. In this study, we investigate allosteric activity regulation for such a class III RNR, the class III RNR from Streptococcus thermophilus(StNrdD). We use a newly developed LC‐MS/MS based activity assay to show that dATP downregulates StNrdD activity and ATP upregulates activity as is true for class Ia RNRs. Using crystallography, we obtain structural data showing that ATP and dATP both bind to the cone domain, and that both the cone domain and GRD are flexible. In particular, in one monomer of StNrdD dimer, the cone domain contacts the GRD, and both domains are ordered, and in the other monomer, the cone domain is positioned away from the GRD and the GRD is disordered. These observations led us to hypothesize that the cone domains of class III RNRs, like those of class Ia RNR, regulate activity through promotion or inhibition of the radical transfer step, but instead of controlling the position of b2, the position of the GRD is allosterically regulated. In this presentation, we show the data described above and show recent cryogenic electron microscopy of ATP‐bound and dATP‐bound states of StNrdD, which reveals differential positioning of the cone domains in the presence of these allosteric effectors. We also present our current thinking about the mechanism of allosteric regulation of activity in a class III RNR.

A suicide diiron oxygenase in p‐aminobenzoate biosynthesis in Chlamydia trachomatis

Folate is an essential one‐carbon carrier cofactor required for essential biological reactions such as amino acid and DNA biosynthesis. Folates are comprised of three chemical moieties: a pteridine ring, p‐aminobenzoate (pABA), and glutamate residues. Chlamydia trachomatis has been shown to synthesize folate de novo; however, it is missing several genes involved in the canonical folate biosynthesis pathway. Previous work has demonstrated that a single gene, ct610, functionally replaces the enzymes responsible for pABA production in the canonical biosynthesis pathway. However, CT610 does not use chorismate as the precursor to pABA as is used in the canonical pathway. Further experiments showed that isotopically labeled tyrosine was incorporated into the pABA molecule when synthesized by CT610 in vivo. However, in vitro experiments demonstrated that CT610 produces pABA in the presence of oxygen and a reducing agent without any added substrates, including tyrosine. This led to the hypothesis that CT610 is a self‐sacrificing “suicide” enzyme that donates an active site tyrosine residue in synthesizing pABA. CT610 has low sequence similarity to non‐heme diiron monooxygenases such as methane monooxygenase and class Ia ribonucleotide reductase, and the previously solved crystal structure of CT610 revealed a diiron active site. Here we describe our recent progress towards understanding this unique suicide mechanism for pABA biosynthesis. To determine the tyrosine residue sacrificed in the reaction, we generated five active site tyrosine to phenylalanine mutants and found that two of the variants completely abolished pABA production, suggesting that one of these residues is the precursor to pABA. Further, a conserved lysine residue is also essential for the reaction and may be a sacrificial amino group donor. We have confirmed the oxygenase activity of wild‐type CT610, which requires only a reducing agent and is not stimulated by the presence of tyrosine or other possible substrates. Further, we developed an Fe(II) reconstitution procedure, where the reconstituted enzyme exhibits a drastic increase in oxygenase activity. However, surprisingly, the reconstituted enzyme does not exhibit increased pABA synthase activity, although EDTA treatment of the as‐purified enzyme almost completely inhibits pABA production. This suggests that a metal other than iron is required for the reaction and thus current work is focused on testing other potential metal cofactors that may play a role in the novel suicide reaction.

Biochemical Characterization of a Human Ribonucleotide Reductase (HsRNR) Variant Lacking Activity Regulation

Ribonucleotide reductase (RNR) is responsible for the synthesis of deoxyribonucleotides used in DNA synthesis and repair. Class Ia RNRs are composed of two dimeric subunits: a catalytic subunit (α2) which contains the active site and two allosteric regulation sites, and radical generating (β2) subunit housing the diferric‐tyrosyl radical cofactor. The two subunits form an α2β2 heterotetrametric complex that allows the radical to travel from β to α subunit for catalysis. RNR activity is regulated by the binding of ATP/dATP to an N‐terminal cone domain. Human RNRs (HsRNRs) are class Ia enzymes that form a homohexameric α6in the presence of either dATP or ATP. Although, both α6 assemblies are morphologically identical, only the α6‐ATP can be disturbed by the addition of β to form the α2β2 complex necessary for catalysis. Recent studies have shown that α subunits contact each other via their cone domains in the α6 rings, but it is unknown if the cone domains contact β2 in the α2β2 active complex. Here, we aim to probe the role of the cone domains in catalysis through biochemical and biophysical studies of an α subunit variant lacking the cone domain. We used site‐directed mutagenesis to delete residues 2‐92 of α, and expressed and purified the variant a protein. The variant protein was used for HPLC‐MS based activity assays of HsRNR. Preliminary results show the cone domain is important for catalysis. Understanding more about RNR’s structure and activity regulation can facilitate development of new cancer therapies.

Ribonucleotide reductase, a novel drug target for gonorrhea.

Antibiotic-resistant Neisseria gonorrhoeae (Ng) are an emerging public health threat due to increasing numbers of multidrug resistant (MDR) organisms. We identified two novel orally active inhibitors, PTC-847 and PTC-672, that exhibit a narrow spectrum of activity against Ng including MDR isolates. By selecting organisms resistant to the novel inhibitors and sequencing their genomes, we identified a new therapeutic target, the class Ia ribonucleotide reductase (RNR). Resistance mutations in Ng map to the N-terminal cone domain of the α subunit, which we show here is involved in forming an inhibited α4β4 state in the presence of the β subunit and allosteric effector dATP. Enzyme assays confirm that PTC-847 and PTC-672 inhibit Ng RNR and reveal that allosteric effector dATP potentiates the inhibitory effect. Oral administration of PTC-672 reduces Ng infection in a mouse model and may have therapeutic potential for treatment of Ng that is resistant to current drugs.

Effects of chameleon dispense-to-plunge speed on particle concentration, complex formation, and final resolution: A case study using the Neisseria gonorrhoeae ribonucleotide reductase inactive complex

Ribonucleotide reductase (RNR) is an essential enzyme that converts ribonucleotides to deoxyribonucleotides and is a promising antibiotic target, but few RNRs have been structurally characterized. We present the use of the chameleon, a commercially-available piezoelectric cryogenic electron microscopy plunger, to address complex denaturation in the Neisseria gonorrhoeae class Ia RNR. Here, we characterize the extent of denaturation of the ring-shaped complex following grid preparation using a traditional plunger and using a chameleon with varying dispense-to-plunge times. We also characterize how dispense-to-plunge time influences the amount of protein sample required for grid preparation and preferred orientation of the sample. We demonstrate that the fastest dispense-to-plunge time of 54 ms is sufficient for generation of a data set that produces a high quality structure, and that a traditional plunging technique or slow chameleon dispense-to-plunge times generate data sets limited in resolution by complex denaturation. The 4.3 Å resolution structure of Neisseria gonorrhoeae class Ia RNR in the inactive α4β4 oligomeric state solved using the chameleon with a fast dispense-to-plunge time yields molecular information regarding similarities and differences to the well studied Escherichia coli class Ia RNR α4β4 ring.

Detection of Catalytically Linked Conformational Changes in Wild-Type Class Ia Ribonucleotide Reductase Using Reaction-Induced FTIR Spectroscopy

The enzyme, ribonucleotide reductase (RNR), is essential for DNA synthesis in all cells. The class Ia Escherichia coli RNR consists of two dimeric subunits, α2 and β2, which form an active but unstable heterodimer of dimers, α2β2. The structure of the wild-type form of the enzyme has been challenging to study due to the instability of the catalytic complex. A long-range proton-coupled electron-transfer (PCET) pathway facilitates radical migration from the Y122 radical-diiron cofactor in the β subunit to an active site cysteine, C439, in the α subunit to initiate the RNR chemistry. The PCET reactions and active site chemistry are spectroscopically masked by a rate-limiting, conformational gate. Here, we present a reaction-induced Fourier transform infrared (RIFTIR) spectroscopic method to monitor the mechanism of the active, wild-type RNR α2β2 complex. This method is employed to obtain new information about conformational changes accompanying RNR catalysis, including the role of carboxylate interactions, deprotonation, and oxidation of active site cysteines, and a detailed description of reversible secondary structural changes. Labeling of tyrosine revealed a conformationally active tyrosine in the β subunit, assigned to Y356β, which is part of the intersubunit PCET pathway. New insights into the roles of the inhibitors, azidoUDP and dATP, and the sensitivity of RIFTIR spectroscopy to detect subtle conformational motions arising from protein allostery are also presented.

Gated Proton Release during Radical Transfer at the Subunit Interface of Ribonucleotide Reductase.

The class Ia ribonucleotide reductase of Escherichia coli requires strict regulation of long-range radical transfer between two subunits, α and β, through a series of redox-active amino acids (Y122•[β] ↔ W48?[β] ↔ Y356[β] ↔ Y731[α] ↔ Y730[α] ↔ C439[α]). Nowhere is this more precarious than at the subunit interface. Here, we show that the oxidation of Y356 is regulated by proton release involving a specific residue, E52[β], which is part of a water channel at the subunit interface for rapid proton transfer to the bulk solvent. An E52Q variant is incapable of Y356 oxidation via the native radical transfer pathway or non-native photochemical oxidation, following photosensitization by covalent attachment of a photo-oxidant at position 355[β]. Substitution of Y356 for various FnY analogues in an E52Q-photoβ2, where the side chain remains deprotonated, recovered photochemical enzymatic turnover. Transient absorption and emission data support the conclusion that Y356 oxidation requires E52 for proton management, suggesting its essential role in gating radical transport across the protein-protein interface.

Subunit Interaction Dynamics of Class Ia Ribonucleotide Reductases: In Search of a Robust Assay.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides (NDP) to deoxynucleotides (dNDP), in part, by controlling the ratios and quantities of dNTPs available for DNA replication and repair. The active form of Escherichia coli class Ia RNR is an asymmetric α2β2 complex in which α2 contains the active site and β2 contains the stable diferric-tyrosyl radical cofactor responsible for initiating the reduction chemistry. Each dNDP is accompanied by disulfide bond formation. We now report that, under in vitro conditions, β2 can initiate turnover in α2 catalytically under both "one" turnover (no external reductant, though producing two dCDPs) and multiple turnover (with an external reductant) assay conditions. In the absence of reductant, rapid chemical quench analysis of a reaction of α2, substrate, and effector with variable amounts of β2 (1-, 10-, and 100-fold less than α2) yields 3 dCDP/α2 at all ratios of α2:β2 with a rate constant of 8-9 s-1, associated with a rate-limiting conformational change. Stopped-flow fluorescence spectroscopy with a fluorophore-labeled β reveals that the rate constants for subunit association (163 ± 7 μM-1 s-1) and dissociation (75 ± 10 s-1) are fast relative to turnover, consistent with catalytic β2. When assaying in the presence of an external reducing system, the turnover number is dictated by the ratio of α2:β2, their concentrations, and the concentration and nature of the reducing system; the rate-limiting step can change from the conformational gating to a step or steps involving disulfide rereduction, dissociation of the inhibited α4β4 state, or both. The issues encountered with E. coli RNR are likely of importance in all class I RNRs and are central to understanding the development of screening assays for inhibitors of these enzymes.

A Proton Wire Mediates Proton Coupled Electron Transfer from Hydroxyurea and Other Hydroxamic Acids to Tyrosyl Radical in Class Ia Ribonucleotide Reductase.

Proton-coupled electron transfer (PCET) is fundamental to many important biological reactions, including solar energy conversion and DNA synthesis. For example, class Ia ribonucleotide reductases (RNRs) contain a tyrosyl radical-diiron cofactor with one aspartate ligand, D84. The tyrosyl radical, Y122•, in the β2 subunit acts as a radical initiator and oxidizes an active site cysteine in the α2 subunit. A transient quaternary α2/β2 complex is induced by substrate and effector binding. The hydroxamic acid, hydroxyurea (HU), reduces Y122• in a PCET reaction involving an electron and proton. This reaction is associated with the loss of activity, a conformational change at Y122, and a change in hydrogen bonding to the Fe1 ligand, D84. Here, we use isotopic labeling, solvent isotope exchange, proton inventories, and reaction-induced Fourier transform infrared (RIFT-IR) spectroscopy to show that the PCET reactions of hydroxamic acids are associated with a characteristic spectrum, which is assignable to electrostatic changes at nonligating aspartate residues. Notably, RIFT-IR spectroscopy reveals this characteristic spectrum when the effects of HU, hydroxylamine, and N-methylhydroxylamine are compared. A large solvent isotope effect is observed for each of the hydroxamic acid reactions, and proton inventories predict that the reactions are associated with the transfer of multiple protons in the transition state. The reduction of Y122• with 4-methoxyphenol does not lead to these characteristic carboxylate shifts and is associated with only a small solvent isotope effect. In addition to studies of the effects of hydroxamic acids on β2 alone, the reactions involving the quaternary α2β2 complex were also investigated. HU treatment of the quaternary complex, α2/β2/ATP/CDP, leads to a similar carboxylate shift spectrum, as observed with β2 alone. The use of globally labeled 13C chimeras (13C α2, 13C β2) confirms the assignment. Because the spectrum is sensitive to 13C β2 labeling, but not 13C α2 labeling, the quaternary complex spectrum is assigned to electrostatic changes in β2 carboxylate groups. Examination of the β2 X-ray structure reveals a hydrogen-bonded network leading from the protein surface to Y122. This predicted network includes nonligating aspartates, glutamate ligands to the iron cluster, and predicted crystallographically resolved water molecules. The network is similar when class Ia RNR structures from Escherichia coli, human, and mouse are compared. We propose that the PCET reactions of hydroxamic acids are mediated by a hydrogen-bonded proton wire in the β2 subunit.

Selenocysteine Substitution in a Class I Ribonucleotide Reductase.

Ribonucleotide reductases (RNRs) employ a complex radical-based mechanism during nucleotide reduction involving multiple active site cysteines that both activate the substrate and reduce it. Using an engineered allo-tRNA, we substituted two active site cysteines with distinct function in the class Ia RNR of Escherichia coli for selenocysteine (U) via amber codon suppression, with efficiency and selectivity enabling biochemical and biophysical studies. Examination of the interactions of the C439U α2 mutant protein with nucleotide substrates and the cognate β2 subunit demonstrates that the endogenous Y122• of β2 is reduced under turnover conditions, presumably through radical transfer to form a transient U439• species. This putative U439• species is formed in a kinetically competent fashion but is incapable of initiating nucleotide reduction via 3'-H abstraction. An analogous C225U α2 protein is also capable of radical transfer from Y122•, but the radical-based substrate chemistry partitions between turnover and stalled reduction akin to the reactivity of mechanism-based inhibitors of RNR. The results collectively demonstrate the essential role of cysteine redox chemistry in the class I RNRs and establish a new tool for investigating thiyl radical reactivity in biology.

High-resolution iron X-ray absorption spectroscopic and computational studies of non-heme diiron peroxo intermediates

Ferritin-like carboxylate-bridged non-heme diiron enzymes activate O2 for a variety of difficult reactions throughout nature. These reactions often begin by abstraction of hydrogen from strong CH bonds. The enzymes activate O2 at their diferrous cofactors to form canonical diferric peroxo intermediates, with a range of possible coordination modes. Herein, we explore the ability of high-energy resolution fluorescence detected X-ray absorption spectroscopy (HERFD XAS) to provide insight into the nature of peroxo level intermediates in non-heme diiron proteins. Freeze quenched (FQ) peroxo intermediates from p-aminobenzoate N-oxygenase (AurF), aldehyde-deformylating oxygenase (ADO), and the β subunit of class Ia ribonucleotide reductase from Escherichia coli (Ecβ) are investigated. All three intermediates are proposed to adopt different peroxo binding modes, and each exhibit different Fe Kα HERFD XAS pre-edge features and intensities. As these FQ-trapped samples consist of multiple species, deconvolution of HERFD XAS spectra based on speciation, as determined by Mössbauer spectroscopy, is also necessitated - yielding ‘pure’ diferric peroxo HERFD XAS spectra from dilute protein samples. Finally, the impact of a given peroxo coordination mode on the HERFD XAS pre-edge energy and intensity is evaluated through time-dependent density functional theory (TDDFT) calculations of the XAS spectra on a series of hypothetical model complexes, which span a full range of possible peroxo coordination modes to a diferric core. The utility of HERFD XAS for future studies of enzymatic intermediates is discussed.

Photochemical Rescue of a Conformationally Inactivated Ribonucleotide Reductase.

Class Ia ribonucleotide reductase (RNR) of Escherichia coli contains an unusually stable tyrosyl radical cofactor in the β2 subunit (Y122•) necessary for nucleotide reductase activity. Upon binding the cognate α2 subunit, loaded with nucleoside diphosphate substrate and an allosteric/activity effector, a rate determining conformational change(s) enables rapid radical transfer (RT) within the active α2β2 complex from the Y122• site in β2 to the substrate activating cysteine residue (C439) in α2 via a pathway of redox active amino acids (Y122[β] ↔ W48[β]? ↔ Y356[β] ↔ Y731[α] ↔ Y730[α] ↔ C439[α]) spanning >35 Å. Ionizable residues at the α2β2 interface are essential in mediating RT, and therefore control activity. One of these mutations, E350X (where X = A, D, Q) in β2, obviates all RT, though the mechanism of control by which E350 mediates RT remains unclear. Herein, we utilize an E350Q-photoβ2 construct to photochemically rescue RNR activity from an otherwise inactive construct, wherein the initial RT event (Y122• → Y356) is replaced by direct photochemical radical generation of Y356•. These data present compelling evidence that E350 conveys allosteric information between the α2 and β2 subunits facilitating conformational gating of RT that specifically targets Y122• reduction, while the fidelity of the remainder of the RT pathway is retained.

Disruption of an oligomeric interface prevents allosteric inhibition of Escherichia coli class Ia ribonucleotide reductase

Ribonucleotide reductases (RNRs) convert ribonucleotides to deoxynucleotides, a process essential for DNA biosynthesis and repair. Class Ia RNRs require two dimeric subunits for activity: an α2 subunit that houses the active site and allosteric regulatory sites and a β2 subunit that houses the diferric tyrosyl radical cofactor. Ribonucleotide reduction requires that both subunits form a compact α2β2 state allowing for radical transfer from β2 to α2 RNR activity is regulated allosterically by dATP, which inhibits RNR, and by ATP, which restores activity. For the well-studied Escherichia coli class Ia RNR, dATP binding to an allosteric site on α promotes formation of an α4β4 ring-like state. Here, we investigate whether the α4β4 formation causes or results from RNR inhibition. We demonstrate that substitutions at the α-β interface (S37D/S39A-α2, S39R-α2, S39F-α2, E42K-α2, or L43Q-α2) that disrupt the α4β4 oligomer abrogate dATP-mediated inhibition, consistent with the idea that α4β4 formation is required for dATP's allosteric inhibition of RNR. Our results further reveal that the α-β interface in the inhibited state is highly sensitive to manipulation, with a single substitution interfering with complex formation. We also discover that residues at the α-β interface whose substitution has previously been shown to cause a mutator phenotype in Escherichia coli (i.e. S39F-α2 or E42K-α2) are impaired only in their activity regulation, thus linking this phenotype with the inability to allosterically down-regulate RNR. Whereas the cytotoxicity of RNR inhibition is well-established, these data emphasize the importance of down-regulation of RNR activity.