Tyrosyl Radical Research Articles

Mechanistic studies of the cofactor assembly in class Ib ribonucleotide reductases and protein affinity for MnII and FeII.

Ribonucleotide reductase (RNR) is an essential enzyme found in all organisms. The function of RNR is to catalyze the conversion of nucleotides to deoxynucleotides. RNRs rely on metallocofactors to oxidize a conserved cysteine in the active site of the enzyme into a thiyl radical, which then initiates nucleotide reduction. The proteins required for MnIII2-Y• cluster formation in class Ib RNRs are NrdF (β-subunit) and NrdI (flavodoxin). An oxidant is channeled from the FMN cofactor in NrdI to the dimanganese center in NrdF, where it oxidizes the dimanganese center and a tyrosyl radical (Y•) is formed. Both Streptococcus sanguinis and Escherichia coli MnII2-NrdF structures have a constriction in the channel immediately above the metal site. In E. coli, the constriction is formed by the side chain of S159, whereas in the S. sanguinis system it involves T158. This serine-to-threonine substitution was investigated using S. sanguinis and Streptococcus pneumoniae class Ib RNRs but it is also present in other pathogenic streptococci. Using stopped-flow kinetics, we investigate the role of this substitution in the mechanism of MnIII2-Y• cluster formation. In addition to different kinetics observed in the studied streptococci, we found that affinity constants of NrdF for MnII and FeII are about 1µM and the previously reported preference for MnII could not be explained by affinity only.

The dynamics of hemoglobin-haptoglobin complexes. Relevance for oxidative stress

Haptoglobin (Hp) is a protein with role in oxidative stress prevention, which it fulfills by trapping hemoglobin (Hb) dimers in a strongly-bound complex, thus reducing Hb's ability to engage in oxidative stress reactions which are greatly enhanced when the Hb tetramer falls apart into dimers. A first molecular dynamics (MD) analysis is presented here for the Hb-Hp pair. Also, for the first time the tyrosyl (Tyr) radicals in globins are examined with MD calculations directed at comparing with electron paramagnetic resonance (EPR)-detected parameters, with the aim of identifying the location of free radicals on the globin. Such assignments are needed in order to complete our understanding of the manner in which globins deal with oxidative stress, as well as for the ways the organism deals with the oxidative stress occasionally accidentally amplified by the globins via their reactive heme and Tyr moieties. The EPR-detected free radical in Hb and in Hb:Hp is characterized experimentally by a measurable increase in hydrophobicity upon Hb:Hp complexation and by a constant ∼53° θ angle. The x-ray structure and MD-derived hydrophobicity-related parameters appear to support Y145, Y35 and Y24 as likely locations of the Hb radical – arguing strongly against Y42, Y130 and Y140. The θ values appear to favor Y42, with the MD data nevertheless also arguing in favor of Y35 and, to a lesser extent, Y145 and Y140. Y130 and Y24 appear the least likely candidates according to the θ values, especially in view of the MD data. Overall, Y35 and Y145 appear to be the two candidates with least arguments against them for satisfying the two criteria (θ values and hydrophobicity) for matching the experimentally-observed EPR radical in Hb and Hb:Hp.

E. coli Ribonucleotide Reductase β2 Subunit Inactivation by Triapine Occurs through Binding of a Triapine-Fe(II) Adduct.

Ribonucleotide reductase (RNR), which supplies the building blocks for DNA biosynthesis and its repair, has been linked to human diseases and is emerging as a therapeutic target. Here, we present a mechanistic investigation of triapine (3AP), a clinically relevant small molecule that inhibits the tyrosyl radical within the RNR β2 subunit. Solvent kinetic isotope effects reveal that proton transfer is not rate-limiting for inhibition of Y122· of E. coli RNR β2 by the pertinent 3AP-Fe(II) adduct. Vibrational spectroscopy further demonstrates that unlike inhibition of the β2 tyrosyl radical by hydroxyurea, a carboxylate containing proton wire is not at play. Binding measurements reveal a low nanomolar affinity (Kd ∼ 6 nM) of 3AP-Fe(II) for β2. Taken together, these data should prompt further development of RNR inactivators based on the triapine scaffold for therapeutic applications.

The three-spin intermediate at the O-O cleavage and proton-pumping junction in heme-Cu oxidases.

Understanding the mechanistic coupling of molecular oxygen reduction and proton pumping for adenosine triphosphate synthesis during cellular respiration is the primary goal of research on heme-copper oxidases—the terminal complex in the membrane-bound electron transport chain. Cleavage of the oxygen-oxygen bond by the heme-copper oxidases forms the key intermediate PM, which initiates proton pumping. This intermediate is now experimentally defined by variable-temperature, variable-field magnetic circular dichroism spectroscopy on a previously unobserved excited state feature associated with its heme iron(IV)-oxo center. These data provide evidence that the iron(IV)-oxo in PM is magnetically coupled to both a copper(II) and a cross-linked tyrosyl radical in the active site. These results provide new insight into the oxygen-oxygen bond cleavage and proton-pumping mechanisms of heme-copper oxidases.

Distribution of H^upbeta Hyperfine Couplings in a Tyrosyl Radical Revealed by 263 GHz ENDOR Spectroscopy

^1H ENDOR spectra of tyrosyl radicals (Y^bullet) have been the subject of numerous EPR spectroscopic studies due to their importance in biology. Nevertheless, assignment of all internal ^1H hyperfine couplings has been challenging because of substantial spectral overlap. Recently, using 263 GHz ENDOR in conjunction with statistical analysis, we could identify the signature of the H^{upbeta _2} coupling in the essential Y_{122} radical of Escherichia coli ribonucleotide reductase, and modeled it with a distribution of radical conformations. Here, we demonstrate that this analysis can be extended to the full-width ^1H ENDOR spectra that contain the larger H^{upbeta _1} coupling. The H^{upbeta _2} and H^{upbeta _1} couplings are related to each other through the ring dihedral and report on the amino acid conformation. The 263 GHz ENDOR data, acquired in batches instead of averaging, and data processing by a new “drift model” allow reconstructing the ENDOR spectra with statistically meaningful confidence intervals and separating them from baseline distortions. Spectral simulations using a distribution of ring dihedral angles confirm the presence of a conformational distribution, consistent with the previous analysis of the H^{upbeta _2} coupling. The analysis was corroborated by 94 GHz ^2H ENDOR of deuterated Y_{122}^bullet. These studies provide a starting point to investigate low populated states of tyrosyl radicals in greater detail.

Titelbild: In‐Cell Characterization of the Stable Tyrosyl Radical in E. coli Ribonucleotide Reductase Using Advanced EPR Spectroscopy (Angew. Chem. 35/2021)

Die Struktur und Regulation des Tyrosyl-Radikals in der Ribonukleotid-Reduktase in E.-coli-Zellen wurde mithilfe moderner EPR-Spektroskopie untersucht. Dieses Radikal startet die Biosynthese von DNA-Bausteinen und ist damit an essentiellen Prozessen des Lebens beteiligt. Die von Müge Kasanmascheff et al. im Forschungsartikel auf S. 19304 vorgestellten Beobachtungen sind überraschend, denn die Befunde deuten auf eine charakteristische Verteilung der Radikale in den Zellen hin, was auf einen komplexen Regulationsmechanismus dieses fundamentalen Enzyms in seiner nativen Umgebung hinweist.

Cover Picture: In‐Cell Characterization of the Stable Tyrosyl Radical in E. coli Ribonucleotide Reductase Using Advanced EPR Spectroscopy (Angew. Chem. Int. Ed. 35/2021)

The structure and regulation of the tyrosyl radical in ribonucleotide reductase in whole E. coli cells was investigated by using advanced EPR spectroscopy. This radical starts the biosynthesis of DNA building blocks, and is thus involved in essential processes in life. The observations reported by Müge Kasanmascheff et al. in their Research Article on page 19155 were surprising and exciting because the findings indicated a distinct radical distribution within the cells, pointing to a complex regulation mechanism of this fundamental enzyme in its native environment.

Molecular insights into the endoperoxide formation by Fe(II)/\u03b1-KG-dependent oxygenase NvfI

Endoperoxide-containing natural products are a group of compounds with structurally unique cyclized peroxide moieties. Although numerous endoperoxide-containing compounds have been isolated, the biosynthesis of the endoperoxides remains unclear. NvfI from Aspergillus novofumigatus IBT 16806 is an endoperoxidase that catalyzes the formation of fumigatonoid A in the biosynthesis of novofumigatonin. Here, we describe our structural and functional analyses of NvfI. The structural elucidation and mutagenesis studies indicate that NvfI does not utilize a tyrosyl radical in the reaction, in contrast to other characterized endoperoxidases. Further, the crystallographic analysis reveals significant conformational changes of two loops upon substrate binding, which suggests a dynamic movement of active site during the catalytic cycle. As a result, NvfI installs three oxygen atoms onto a substrate in a single enzyme turnover. Based on these results, we propose a mechanism for the NvfI-catalyzed, unique endoperoxide formation reaction to produce fumigatonoid A.

Müge Kasanmascheff.

"Science is fun because it is full of surprises … My science 'heroes' are JoAnne Stubbe and Marina Bennati …" Find out more about Müge Kasanmascheff in her Introducing … Profile.

Müge Kasanmascheff

Müge Kasanmascheff

Evidence for the Mn4-Yz[rad] Magnetic Interaction in Ca2+- depleted Photosystem II

The Photosystem II (PSII) water oxidation to molecular oxygen process is catalyzed by an oxygen-bridged Mn4CaO5 cluster, known as Oxygen Evolving Complex (OEC) of PSII. The Mn4CaO5 cluster undergoes periodically four one-electron oxidation steps, S0 → S1, S1 → S2, S2 → S3, S3 → S0. The O2 release takes place during the S3-[S4]-S0 transition, S4 being a transient. The intermediates of the S-state transitions are known as metalloradical intermediate states (SiYz) and involve the free radical of Tyrosine Z (Yz, TyrZ). In most metalloradical states it was established that Yz interacts magnetically with the Mn4CaO5 cluster. However, in Ca2+- depleted PSII preparations the spin-spin interaction between the Mn4O5 and Yz, during the formation of the S2Yz intermediate state was not strongly supported experimentally. In our effort to investigate the existence of the aforementioned interaction, we took advantance of the NIR sensitivity of the S2 state of Mn4 and examined whether the S2Yz EPR spectrum can be modified. Our EPR experiments, combined with the simulation analysis of the spectra show that reversible modification of the S2 EPR spectrum by NIR irradiation results in a reversible change of the Yz EPR signal shape. These observations strongly support the idea of the magnetic interaction between the Mn4 and tyrosyl radical, upon the formation of the S2Yz metalloradical state in Ca2+- depleted PSII membranes.

Influence of pH on radical reactions between kynurenic acid and amino acids tryptophan and tyrosine. Part I. Amino acids in free state

In the human eye lens the endogenous chromophores of UV-A light (315–400 nm) are able to sensitize radical reactions leading to protein modifications during normal aging and the cataract progression. Kynurenic acid (KNA−) is the most photochemically active dye of the human eye lens reported to date with pKa(KNAH2•) 5.5 for its radical form. Cataract is thought to develop under oxidative stress which could be accompanied by acidosis, an acidification of the intracellular environment. Protonation of kynurenyl radicals at mildly acidic conditions may change the outcome of radical reactions leading to additional damage to proteins. In this work we investigated the influence of pH on the degradation of initial reagents and the formation of products in photoinduced radical reactions between KNA− and amino acids tryptophan (Trp) and tyrosine (Tyr) in free states. Our results have shown that pH variation has minor influence on kinetics of reagent decay and accumulation of products in reactions between tyrosyl and kynurenic acid radicals. However in the case of Trp a two-fold decrease of the reagent degradation without visible changes in the composition of formed products was observed with pH decrease from 7 to 3. Time-resolved measurements have shown similar acidification-induced two-fold acceleration of decay of kynurenyl and tryptophanyl radicals via Back Electron Transfer (BET) with the restoration of initial reagents. Experiments with tryptophan derivatives with different pKa values for their radical forms point out the protonation of tryptophanyl radical as the driving force for BET acceleration at low pH. Our results demonstrate that the protonation of kynurenyl radical does not change its reactivity towards amino acids radicals but the total yield of radical photodamage decreases with the protonation of tryptophanyl radicals. It could be expected that radical induced damage to proteins will depend on the pKa of tryptophanyl radicals within a protein globule.

Coumarin-Based Triapine Derivatives and Their Copper(II) Complexes: Synthesis, Cytotoxicity and mR2 RNR Inhibition Activity.

A series of thiosemicarbazone-coumarin hybrids (HL1-HL3 and H2L4) has been synthesised in 12 steps and used for the preparation of mono- and dinuclear copper(II) complexes, namely Cu(HL1)Cl2 (1), Cu(HL2)Cl2 (2), Cu(HL3)Cl2 (3) and Cu2(H2L4)Cl4 (4), isolated in hydrated or solvated forms. Both the organic hybrids and their copper(II) and dicopper(II) complexes were comprehensively characterised by analytical and spectroscopic techniques, i.e., elemental analysis, ESI mass spectrometry, 1D and 2D NMR, IR and UV–vis spectroscopies, cyclic voltammetry (CV) and spectroelectrochemistry (SEC). Re-crystallisation of 1 from methanol afforded single crystals of copper(II) complex with monoanionic ligand Cu(L1)Cl, which could be studied by single crystal X-ray diffraction (SC-XRD). The prepared copper(II) complexes and their metal-free ligands revealed antiproliferative activity against highly resistant cancer cell lines, including triple negative breast cancer cells MDA-MB-231, sensitive COLO-205 and multidrug resistant COLO-320 colorectal adenocarcinoma cell lines, as well as in healthy human lung fibroblasts MRC-5 and compared to those for triapine and doxorubicin. In addition, their ability to reduce the tyrosyl radical in mouse R2 protein of ribonucleotide reductase has been ascertained by EPR spectroscopy and the results were compared with those for triapine.

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.

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

AbstractThe 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.

The Redox-Active Tyrosine Is Essential for Proton Pumping in Cytochrome c Oxidase

Cellular respiration involves electron transport via a number of enzyme complexes to the terminal Cytochrome c oxidase (CcO), in which molecular oxygen is reduced to water. The free energy released in the reduction process is used to establish a transmembrane electrochemical gradient, via two processes, both corresponding to charge transport across the membrane in which the enzymes are embedded. First, the reduction chemistry occurring in the active site of CcO is electrogenic, which means that the electrons and protons are delivered from opposite sides of the membrane. Second, the exergonic chemistry is coupled to translocation of protons across the entire membrane, referred to as proton pumping. In the largest subfamily of the CcO enzymes, the A-family, one proton is pumped for every electron needed for the chemistry, making the energy conservation particularly efficient. In the present study, hybrid density functional calculations are performed on a model of the A-family CcOs. The calculations show that the redox-active tyrosine, conserved in all types of CcOs, plays an essential role for the energy conservation. Based on the calculations a reaction mechanism is suggested involving a tyrosyl radical (possibly mixed with tyrosinate character) in all reduction steps. The result is that the free energy released in each reduction step is large enough to allow proton pumping in all reduction steps without prohibitively high barriers when the gradient is present. Furthermore, the unprotonated tyrosine provides a mechanism for coupling the uptake of two protons per electron in every reduction step, i.e. for a secure proton pumping.

The flavodoxin FldA activates the class Ia ribonucleotide reductase of Campylobacter jejuni.

Campylobacter jejuni is a microaerophilic zoonotic pathogen with an atypical respiratory Complex I that oxidizes a flavodoxin (FldA) instead of NADH. FldA is essential for viability and is reduced via pyruvate and 2-oxoglutarate oxidoreductases (POR/OOR). Here, we show that FldA can also be reduced by FqrB (Cj0559), an NADPH:FldA reductase. An fqrB deletion mutant was viable but displayed a significant growth defect. FqrB is related to flavoprotein reductases from Gram-positive bacteria that can reduce NrdI, a specialized flavodoxin that is needed for tyrosyl radical formation in NrdF, the beta subunit of class 1b-type (Mn) ribonucleotide reductase (RNR). However, C.jejuni possesses a single class Ia-type (Fe) RNR (NrdAB) that would be expected to be ferredoxin dependent. We show that CjFldA is an unusually high potential flavodoxin unrelated to NrdI, yet growth of the fqrB mutant, but not the wild-type or a complemented strain, was stimulated by low deoxyribonucleoside (dRNS) concentrations, suggesting FldA links FqrB and RNR activity. Using purified proteins, we confirmed the NrdB tyrosyl radical could be regenerated in an NADPH, FqrB, and FldA dependent manner, as evidenced by both optical and electron paramagnetic resonance (EPR) spectroscopy. Thus, FldA activates RNR in C.jejuni, partly explaining its essentiality.

On the Track of Long-Range Electron Transfer in B-Type Dye-Decolorizing Peroxidases: Identification of a Tyrosyl Radical by Computational Prediction and Electron Paramagnetic Resonance Spectroscopy.

The catalytic activity of dye-decolorizing peroxidases (DyPs) toward bulky substrates, including anthraquinone dyes, phenolic lignin model compounds, or 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), is in strong contrast to their sterically restrictive active site. In two of the three known subfamilies (A- and C/D-type DyPs), catalytic protein radicals at surface-exposed sites, which are connected to the heme cofactor by electron transfer path(s), have been identified. So far in B-type DyPs, there has been no evidence for protein radical formation after activation by hydrogen peroxide. Interestingly, B-type Klebsiella pneumoniae dye-decolorizing peroxidase (KpDyP) displays a persistent organic radical in the resting state composed of two species that can be distinguished by W-band electron spin echo electron paramagnetic resonance (EPR) spectroscopy. Here, on the basis of a comprehensive mutational and EPR study of computationally predicted tyrosine and tryptophan variants of KpDyP, we demonstrate the formation of tyrosyl radicals (Y247 and Y92) and a radical-stabilizing Y-W dyad between Y247 and W18 in KpDyP, which are unique to enterobacterial B-type DyPs. Y247 is connected to Y92 by a hydrogen bonding network, is solvent accessible in simulations, and is involved in ABTS oxidation. This suggests the existence of long-range electron path(s) in B-type DyPs. The mechanistic and physiological relevance of the reaction mechanism of B-type DyPs is discussed.

Sensitized photo-oxidation of plant cytokinin-specific binding protein - Does the environment of the thioether group influence the oxidation reaction? From primary intermediates to stable products

The reactions of protein oxidation play a significant role in many biological processes, especially in diseases development. Therefore, it is important to understand, how the protein molecule behaves in the presence of oxidants. In the present work, photo-oxidation of phytohormone-binding plant protein (VrPhBP) was investigated using light and 3-carboxybenzophenone (3CB) as a sensitizer (one electron oxidant).The protein interacts with the sensitizer in the ground state forming a weak binding complex leading to the presence of bound and free 3CB in solution. The early events and transient species (such as radicals and radical ions) formed during irradiation were characterised by transient spectroscopy showing the formation of the sulphur radical cation Met>S●+ (stabilized by (S∴N)+)and the tyrosyl radical TyrO● on VrPhBP. Thus the 3CB excited triplet state was quenched by the Met and Tyr residues and mostly by Met (based on the deconvoluted transient absorption spectra).The presence of a Tyr side chain in the vicinity of a Met residue results in intramolecular electron transfer from Tyr to the Met>S●+ radical cation, leading to regeneration of the thioether side chain and formation of TyrO●. The presence of other side chains close to Met, such as Arg or Lys can induce the stabilization of Met>S●+ via the formation of two-centered three-electron bonded species (S∴N)+.The transient species were additionally confirmed by stable product analysis. Based on SDS-PAGE, chromatography and mass spectrometry, the formation of methionine sulphoxide and Met-3CB adduct was identified together with di-Tyr cross links. On the basis of the experimental results the overall mechanism of VrPhBP photo-oxidation, from its early events to the formation of stable products, is described. In addition, a good correlation between the mechanisms of photooxidation of model compounds such as Met derivatives and peptides and those for real biological systems is emphasized.