Coli Ribonucleotide Reductase Research Articles

In-Cell Characterization of the Stable Tyrosyl Radical in E. coli Ribonucleotide Reductase Using Advanced EPR Spectroscopy.

The E. coli ribonucleotide reductase (RNR), a paradigm for class Ia enzymes including human RNR, catalyzes the biosynthesis of DNA building blocks and requires a di‐iron tyrosyl radical (Y122 .) cofactor for activity. The knowledge on the in vitro Y122 . structure and its radical distribution within the β2 subunit has accumulated over the years; yet little information exists on the in vivo Y122 .. Here, we characterize this essential radical in whole cells. Multi‐frequency EPR and electron‐nuclear double resonance (ENDOR) demonstrate that the structure and electrostatic environment of Y122 . are identical under in vivo and in vitro conditions. Pulsed dipolar EPR experiments shed light on a distinct in vivo Y122 . per β2 distribution, supporting the key role of Y. concentrations in regulating RNR activity. Additionally, we spectroscopically verify the generation of an unnatural amino acid radical, F3Y122 ., in whole cells, providing a crucial step towards unique insights into the RNR catalysis under physiological conditions.

Detection of Water Molecules on the Radical Transfer Pathway of Ribonucleotide Reductase by 17O Electron-Nuclear Double Resonance Spectroscopy.

The role of water in biological proton-coupled electron transfer (PCET) is emerging as a key for understanding mechanistic details at atomic resolution. Here we demonstrate 17O high-frequency electron–nuclear double resonance (ENDOR) in conjunction with H217O-labeled protein buffer to establish the presence of ordered water molecules at three radical intermediates in an active enzyme complex, the α2β2E. coli ribonucleotide reductase. Our data give unambiguous evidence that all three, individually trapped, intermediates are hyperfine coupled to one water molecule with Tyr-O···17O distances in the range 2.8–3.1 Å. The availability of this structural information will allow for quantitative models of PCET in this prototype enzyme. The results also provide a spectroscopic signature for water H-bonded to a tyrosyl radical.

Finding the Missing Link - Modeling the Conformational Space of the Active and Inactive forms of E. Coli Ribonucleotide Reductase

Ribonucleotide Reductase (RNR) is an enzyme that catalyzes the production of deoxyribonucletides from ribonucleotides. The active form of E. coli RNR is comprised of two homodimeric subunits, α2 and β2, and catalysis involves radical transfer between Y122 on the β2 subunit and C439 on the α2 subunit. By contrast, recent structural data suggest that the dATP-inhibited (inactive) form of the enzyme corresponds to an α4β4 ring. In this structure Y122 on the β2 subunit and C439 on the α2 subunit are separated by more than 60 angstroms - a finding that explains, in part, why this structure is inactive. Interestingly, in crystal structures of the inactive complex, ∼27 residues near the C-terminus the β2 subunit do not have electron density, suggesting that these residues are disordered in the inactive form of the enzyme. This missing “linker region” contains a tyrosine residue (Y356) that plays a crucial role in radical transfer in the active complex. In this work we use molecular simulations to explore the conformational space of these disordered residues in both active and inactive models of E. coli RNR. Our data suggest that Y356 adopts states that are inconsistent with radical transfer in the inactive complex. These observations argue that inactivity is explained both by the increased distance required for radical transfer, and by the relative position of crucial residues in the flexible linker region in the inactive state.

Photoactive Peptides for Light-Initiated Tyrosyl Radical Generation and Transport into Ribonucleotide Reductase

The mechanism of radical transport in the alpha2 (R1) subunit of class I E. coli ribonucleotide reductase (RNR) has been investigated by the phototriggered generation of a tyrosyl radical, *Y356, on a 20-mer peptide bound to alpha2. This peptide, Y-R2C19, is identical to the C-terminal peptide tail of the beta2 (R2) subunit and is a known competitive inhibitor of binding of the native beta2 protein to alpha2. *Y356 radical initiation is prompted by excitation (lambda >or= 300 nm) of a proximal anthraquinone, Anq, or benzophenone, BPA, chromophore on the peptide. Transient absorption spectroscopy has been employed to kinetically characterize the radical-producing step by time resolving the semiquinone anion (Anq*-), ketyl radical (*-BPA), and Y* photoproducts on (i) BPA-Y and Anq-Y dipeptides and (ii) BPA/Anq-Y-R2C19 peptides. Light-initiated, single-turnover assays have been carried out with the peptide/alpha2 complex in the presence of [14C]-labeled cytidine 5'-diphosphate substrate and ATP allosteric effector. We show that both the Anq- and BPA-containing peptides are competent in deoxycytidine diphosphate formation and turnover occurs via Y731 to Y730 to C439 pathway-dependent radical transport in alpha2. Experiments with the Y730F mutant exclude a direct superexchange mechanism between C439 and Y731 and are consistent with a PCET model for radical transport in which there is a unidirectional transport of the electron and proton transport among residues of alpha2.

EPR Distance Measurements Support a Model for Long-Range Radical Initiation in E. coli Ribonucleotide Reductase

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

Retrostructural analysis of metalloproteins: application to the design of a minimal model for diiron proteins.

De novo protein design provides an attractive approach for the construction of models to probe the features required for function of complex metalloproteins. The metal-binding sites of many metalloproteins lie between multiple elements of secondary structure, inviting a retrostructural approach to constructing minimal models of their active sites. The backbone geometries comprising the metal-binding sites of zinc fingers, diiron proteins, and rubredoxins may be described to within approximately 1 A rms deviation by using a simple geometric model with only six adjustable parameters. These geometric models provide excellent starting points for the design of metalloproteins, as illustrated in the construction of Due Ferro 1 (DF1), a minimal model for the Glu-Xxx-Xxx-His class of dinuclear metalloproteins. This protein was synthesized and structurally characterized as the di-Zn(II) complex by x-ray crystallography, by using data that extend to 2.5 A. This four-helix bundle protein is comprised of two noncovalently associated helix-loop-helix motifs. The dinuclear center is formed by two bridging Glu and two chelating Glu side chains, as well as two monodentate His ligands. The primary ligands are mostly buried in the protein interior, and their geometries are stabilized by a network of hydrogen bonds to second-shell ligands. In particular, a Tyr residue forms a hydrogen bond to a chelating Glu ligand, similar to a motif found in the diiron-containing R2 subunit of Escherichia coli ribonucleotide reductase and the ferritins. DF1 also binds cobalt and iron ions and should provide an attractive model for a variety of diiron proteins that use oxygen for processes including iron storage, radical formation, and hydrocarbon oxidation.

New Paramagnetic Species Formed at the Expense of the Transient Tyrosyl Radical in Mutant Protein R2 F208Y ofEscherichia coliRibonucleotide Reductase

The highly conserved residue F208 in protein R2 ofE. coliribonucleotide reductase is close to the binuclear iron center, and found to be involved in stabilizing the tyrosyl radical Y122• in wild type R2. Upon the reconstitution reaction of the mutant R2 F208Y with ferrous iron and molecular oxygen, we observed a new EPR singlet signal (g=2.003) formed concomitantly with decay of the transient tyrosyl radical Y122• (g=2.005). This new paramagnetic species (denoted Z) was stable for weeks at 4°C and visible by EPR only below 50 K. The EPR singlet could not be saturated by available microwave power, suggesting that Z may be a mainly metal centered species. The maximum amount of the compound Z in the protein purified from cells grown in rich medium was about 0.18 unpaired spin/R2. An identical EPR signal of Z was found also in the double mutant R2 F208Y/Y122F. In the presence of high concentration of sodium ascorbate, the amounts of both the transient Y122• and the new species Z increased considerably in the reconstitution reaction. The results suggest that Z is most likely an oxo-ferryl species possibly in equilibrium with a Y208 ligand radical.

Differentiation of Six Distinct Thioredoxins in Seeds of the Soybean

The thioredoxin profile of non-photosynthetic plant tissues, in particular of seeds, is much less known than that of green leaves. We have systematically fractionated and assayed the low molecular weight, heat-stable proteins present in extracts from soybean seeds by a variety of chromatographic and enzymatic methods. A multiplicity of six thioredoxins has been established, closely comparable to the pattern previously observed in soybean leaves (Häberlein,1991). Purification to homogeneity of the six proteins was achieved by FPLC. Thioredoxin activities in vitro towards soybean chloroplast NADP-MDH, fructosebisphosphatase, NADPH thioredoxin reductase, and E. coli ribonucleotide reductase have been measured. Some of the seed thioredoxins exhibit higher molecular weights, stronger binding to anion exchange columns, and much lower specificity in enzyme assays than their counterparts isolated from leaves, suggesting the presence of unspecialized storage forms in the dry seed.

Electron Paramagnetic Resonance Study of the Mixed-Valent Diiron Center in Escherichia coli Ribonucleotide Reductase Produced by Reduction of Radical-Free Protein R2 at 77 K

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTElectron Paramagnetic Resonance Study of the Mixed-Valent Diiron Center in Escherichia coli Ribonucleotide Reductase Produced by Reduction of Radical-Free Protein R2 at 77 KRoman Davydov, Sergei Kuprin, Astrid Graeslund, and Anders EhrenbergCite this: J. Am. Chem. Soc. 1994, 116, 24, 11120–11128Publication Date (Print):November 1, 1994Publication History Published online1 May 2002Published inissue 1 November 1994https://doi.org/10.1021/ja00103a029RIGHTS & PERMISSIONSArticle Views297Altmetric-Citations82LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts

Mechanism of Assembly of the Tyrosyl Radical-Diiron(III) Cofactorof E. coli Ribonucleotide Reductase. 3. Kinetics of the Limiting Fe2+ Reaction by Optical, EPR, and Moessbauer Spectroscopies

Mechanism of Assembly of the Tyrosyl Radical-Diiron(III) Cofactorof E. coli Ribonucleotide Reductase. 3. Kinetics of the Limiting Fe2+ Reaction by Optical, EPR, and Moessbauer Spectroscopies

Mechanism of Assembly of the Tyrosyl Radical-Diiron(III) Cofactor of E. Coli Ribonucleotide Reductase: 1. Moessbauer Characterization of the Diferric Radical Precursor

Mechanism of Assembly of the Tyrosyl Radical-Diiron(III) Cofactor of E. Coli Ribonucleotide Reductase: 1. Moessbauer Characterization of the Diferric Radical Precursor

Crystallization and preliminary structure analysis of protein R2 mutants ofEscherichia coliribonucleotide reductase

Crystallization and preliminary structure analysis of protein R2 mutants of<i>Escherichia coli</i>ribonucleotide reductase

A Mössbauer investigation of the kinetic intermediates involved in formation of the tyrosyl radical-diferric center cofactor in E. Coli ribonucleotide reductase

A Mössbauer investigation of the kinetic intermediates involved in formation of the tyrosyl radical-diferric center cofactor in E. Coli ribonucleotide reductase

Ribonucleoside Diphosphate Reductase Induced by Bacteriophage T4: I. PURIFICATION AND CHARACTERIZATION

Abstract Ribonucleoside diphosphate reductase induced in Escherichia coli after infection with bacteriophage T4 was prepared in an essentially pure state by a method involving among other things affinity chromatography on dATP-Sepharose. The enzyme has a molecular weight of approximately 225,000 as determined by a combination of gel filtration and velocity sedimentation techniques. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicates the presence of equimolar amounts of two polypeptide chains, α and β, with molecular weights of about 85,000 and 35,000, respectively. It is suggested that the native enzyme has the subunit structure α2β2. This structure is analogous to that of the active form of E. coli ribonucleotide reductase. α2 corresponds in size to protein B1 and β2 to protein B2. In contrast to the bacterial enzyme the phage enzyme does not dissociate into α2 and β2 on purification. T4 ribonucleotide reductase contains 2.3 atoms of non-heme iron per molecule. The light absorption spectrum shows a sharp peak at 410 nm, and the electron spin resonance spectrum shows a doublet structure centered at g = 2.00. Both spectra are very similar to those of protein B2 of the E. coli ribonucleotide reductase. The reduction of ribonucleoside diphosphates shows absolute requirements for NADPH, T4 thioredoxin, and E. coli thioredoxin reductase. The reaction is stimulated 2- to 3-fold by MgCl2.