Amino Acid Radicals Research Articles

2.6-Å resolution cryo-EM structure of a class Ia ribonucleotide reductase trapped with mechanism-based inhibitor N3CDP

Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides using radical-based chemistry. For class Ia RNRs, the radical species is stored in a separate subunit (β2) from the subunit housing the active site (α2), requiring the formation of a short-lived α2β2 complex and long-range radical transfer (RT). RT occurs via proton-coupled electron transfer (PCET) over a long distance (~32-Å) and involves the formation and decay of multiple amino acid radical species. Here, we use cryogenic electron microscopy and a mechanism-based inhibitor 2'-azido-2'-deoxycytidine-5'-diphosphate (N3CDP) to trap a wild-type α2β2 complex of Escherichia coli class Ia RNR. We find that one α subunit has turned over and that the other is trapped, bound to β in a midturnover state. Instead of N3CDP in the active site, forward RT has resulted in N2 loss, migration of the third nitrogen from the ribose C2' to C3' positions, and attachment of this nitrogen to the sulfur of cysteine-225. In this study, an inhibitor has been visualized as an adduct to an RNR. Additionally, this structure reveals the positions of PCET residues following forward RT, complementing the previous structure that depicted a preturnover PCET pathway and suggesting how PCET is gated at the α-β interface. This N3CDP-trapped structure is also of sufficient resolution (2.6 Å) to visualize water molecules, allowing us to evaluate the proposal that water molecules are proton acceptors and donors as part of the PCET process.

Intermolecular hydrogen bonding behavior of amino acid radical cations.

Amino acid and peptide radicals are of broad interest due to their roles in biochemical oxidative damage, pathogenesis and protein radical catalysis, among others. Using density functional theory (DFT) calculations at the ωB97X-D/def2-QZVPPD//ωB97X-D/def2-TZVPP level of theory, we systematically investigated the hydrogen bonding between water and fourteen α-amino acids (Ala, Asn, Cys, Gln, Gly, His, Met, Phe, Pro, Sel, Ser, Thr, Trp, and Tyr) in both neutral and radical cation forms. For all amino acids surveyed, stronger hydrogen-bonding interactions with water were observed upon single-electron oxidation, with the greatest increases in hydrogen-bonding strength occurring in Gly, Ala and His. We demonstrate that the side chain has a significant impact on the most favorable hydrogen-bonding modes experienced by amino acid radical cations. Our computations also explored the fragmentation of amino acid radical cations through the loss of a COOH radical facilitated by hydrogen bonding. The most favorable pathways provided stabilization of the resulting cationic fragments through hydrogen bonding, resulting in more favorable thermodynamics for the fragmentation process. These results indicate that non-covalent interactions with the environment have a profound impact on the structure and chemical fate of oxidized amino acids.

The Mechanism of Inhibition of Pyruvate Formate Lyase by Methacrylate.

Pyruvate Formate Lyase (PFL) catalyzes acetyl transfer from pyruvate to coenzyme a by a mechanism involving multiple amino acid radicals. A post-translationally installed glycyl radical (G734· in Escherichia coli) is essential for enzyme activity and two cysteines (C418 and C419) are proposed to form thiyl radicals during turnover, yet their unique roles in catalysis have not been directly demonstrated with both structural and electronic resolution. Methacrylate is an isostructural analog of pyruvate and an informative irreversible inhibitor of pfl. Here we demonstrate the mechanism of inhibition of pfl by methacrylate. Treatment of activated pfl with methacrylate results in the conversion of the G734· to a new radical species, concomitant with enzyme inhibition, centered at g = 2.0033. Spectral simulations, reactions with methacrylate isotopologues, and Density Functional Theory (DFT) calculations support our assignment of the radical to a C2 tertiary methacryl radical. The reaction is specific for C418, as evidenced by mass spectrometry. The methacryl radical decays over time, reforming G734·, and the decay exhibits a H/D solvent isotope effect of 3.4, consistent with H-atom transfer from an ionizable donor, presumably the C419 sulfhydryl group. Acrylate also inhibits PFL irreversibly, and alkylates C418, but we did not observe an acryl secondary radical in H2O or in D2O within 10 s, consistent with our DFT calculations and the expected reactivity of a secondary versus tertiary carbon-centered radical. Together, the results support unique roles of the two active site cysteines of PFL and a C419 S-H bond dissociation energy between that of a secondary and tertiary C-H bond.

Natural and synthetic peptides in antimicrobial therapy

Antimicrobial function of innate immunity is mediated by the low-molecular weight peptides which are active against bacteria, fungi and some viruses. The review presents data on studies of both natural and synthetic peptides regarding the features of their structure and therapeutic effect. As a rule, the molecules of such peptides are positively charged, due to amino acid radicals capable of protonation. Spatially, antimicrobial peptide molecules are arranged as -helices or -layers in separate or compound assemblies. At the same time, short molecular chains, including up to 18 amino acid residues, exist as a linear or cyclic forms, remaining at the level of primary spatial structure. Natural antimicrobial peptides are predominantly produced by neutrophilic granulocytes and macrophages, as well as epithelial cells of the barrier organs. Three families of natural antimicrobial peptides have been most studied: defensins, cathelicidins, and histatins. Defensins are active against Gram-positive and Gram-negative bacteria, viruses and fungi, having anti-inflammatory and immunomodulatory activity. Cathelicidins are chemoattractants and exert antibacterial, immunomodulatory, wound healing, antitumor effects, potentially contributing to the development of autoimmune diseases. Histatins have a pronounced fungicidal effect and prevent the formation of bacterial biofilms. A detailed study on the structure and principles of action of natural antimicrobial peptides made it possible to apply this information for the in vitro synthesis of peptides thus making it possible to create multipurpose drugs based on them. E.g., synthetic peptides WR12 and D-IK8 ensure the delivery of antibiotics to infected or tumor cells, due to permeabilization of cellular membranes. At the same time, a synthetic peptide, acipensin 1, is capable of penetrating into human tumor cells without damaging them. The immunomodulatory peptide glutoxim is effectively used in anti-tuberculosis therapy. ZP2 peptide, the functional site of granulocyte-macrophage colony-stimulating factor is effective against Gram-negative bacteria (K. pneumoniae, P. aeruginosa and A. baumannii) as well as EpsteinBarr virus. Thymic immunoregulatory peptides bestim, hepon, thymogen and imunofan are inducers of endogenous - and -interferon production, inhibit the development of malignant neoplasms, and possess anti-inflammatory activity. Gepon is used in the treatment of viral hepatitis, respiratory and opportunistic infections, croup syndrome and sexually transmitted infections (including genital herpes). Thus, the synthetic antimicrobial peptides are widely used in complex treatment regimens along with conventional antibiotics, antiviral, and antitumor drugs, thus making it possible to achieve higher therapeutic effect.

Oxidative stress and protection against it in bacteria

Microorganisms are exposed to reactive oxygen species (ROS) that are formed in various ways, in particular, as a result of respiration or other intracellular processes, during metal-catalyzed Fenton reactions, as a result of the action of UV- and X-radiation, under the influence of some antimicrobial drugs, or during the host immune oxidative-burst response against infection agents. In this review, we take a look at the mechanisms of microbial cell damage, including damage of lipids and proteins. Lipid peroxidation (LPO) is one of the main molecular mechanisms involved in oxidative damage to cellular structures. A variety of products are formed during LPO reactions: alkoxyl radicals, peroxyl radicals, hydroperoxides, diene conjugates, carbonyl compounds, aldehyde adducts with biopolymers, alcohols, esters, etc. These products include cytotoxic and highly reactive compounds. Free radical reactions of protein damage occur via hydrogen atom abstraction from α-carbon or SH-, NH2-groups of aminoacids and electron abstraction from nucleophile centers of proteins resulting in the fragmentation of proteins, their denaturation and the formation of amino acid radicals. Bacteria show a significant adaptive potential to the influence of stress agents, including ROS. We summarized the data on bacterial antioxidant protection, ROS redox sensors, and regulators of bacterial cell response to ROS exposure, focusing on the features of anaerobic microorganisms, as their responses to the oxidative damage are the least studied, and many problems remain unsolved. This review contains information about changes in fatty acid composition of lipids of the plasma membrane to maintain the necessary fluidity, and, thus, counteract the effects of various stressing agents, including ROS. The main modifications of the fatty acid composition of lipids important for the regulation of membrane fluidity are described, in particular, via changes in the degree of lipid saturation, cis/trans isomerization, and synthesis of cyclopropane fatty acids.

Amino Acid Sulfinate Salts as Alkyl Radical Precursors

A general approach to the synthesis of amino acid sulfinate salts from commercially available α-chiral hydroxylated amino acids is reported. These reagents are shown to be valuable precursors to alkyl radicals under mild photochemical oxidation conditions. The photochemically generated amino acid radicals engage readily with alkyl and aryl disulfide radical traps to afford a diverse suite of modified amino acids.

Stereoretentive Post-Translational Protein Editing.

Chemical post-translational methods allow convergent side-chain editing of proteins without needing to resort to genetic intervention. Current approaches that allow the creation of constitutionally native side chains via C-C bond formation, using off-protein carbon-centered C· radicals added to unnatural amino acid radical acceptor (SOMOphile, singly occupied molecular orbital (SOMO)) "tags" such as dehydroalanine, are benign and wide-ranging. However, they also typically create epimeric mixtures of d/l-residues. Here, we describe a light-mediated desulfurative method that, through the creation and reaction of stereoretained on-proteinl-alanyl Cβ· radicals, allows Cβ-Hγ, Cβ-Oγ, Cβ-Seγ, Cβ-Bγ, and Cβ-Cγ bond formation to flexibly generate site-selectively edited proteins with full retention of native stereochemistry under mild conditions from a natural amino acid precursor. This methodology shows great potential to explore protein side-chain diversity and function and in the construction of useful bioconjugates.

Compound I Formation and Reactivity in Dimeric Chlorite Dismutase: Impact of pH and the Dynamics of the Catalytic Arginine.

The heme enzyme chlorite dismutase (Cld) catalyzes the degradation of chlorite to chloride and dioxygen. Many questions about the molecular reaction mechanism of this iron protein have remained unanswered, including the electronic nature of the catalytically relevant oxoiron(IV) intermediate and its interaction with the distal, flexible, and catalytically active arginine. Here, we have investigated the dimeric Cld from Cyanothece sp. PCC7425 (CCld) and two variants having the catalytic arginine R127 (i) hydrogen-bonded to glutamine Q74 (wild-type CCld), (ii) arrested in a salt bridge with a glutamate (Q74E), or (iii) being fully flexible (Q74V). Presented stopped-flow spectroscopic studies demonstrate the initial and transient appearance of Compound I in the reaction between CCld and chlorite at pH 5.0 and 7.0 and the dominance of spectral features of an oxoiron(IV) species (418, 528, and 551 nm) during most of the chlorite degradation period at neutral and alkaline pH. Arresting the R127 in a salt bridge delays chlorite decomposition, whereas increased flexibility accelerates the reaction. The dynamics of R127 does not affect the formation of Compound I mediated by hypochlorite but has an influence on Compound I stability, which decreases rapidly with increasing pH. The decrease in activity is accompanied by the formation of protein-based amino acid radicals. Compound I is demonstrated to oxidize iodide, chlorite, and serotonin but not hypochlorite. Serotonin is able to dampen oxidative damage and inactivation of CCld at neutral and alkaline pH. Presented data are discussed with respect to the molecular mechanism of Cld and the pronounced pH dependence of chlorite degradation.

Roles of High-valent Hemes and pH Dependence in Halite Decomposition Catalyzed by Chlorite Dismutase from Dechloromonas aromatica.

The heme-based chlorite dismutases catalyze the unimolecular decomposition of chlorite (ClO2 -) to yield Cl- and O2. The work presented here shows that chlorite dismutase from Dechloromonas aromatica (DaCld) also catalyzes the decomposition of bromite (BrO2 -) with the evolution of O2 (k cat = (2.0±0.2)×102 s-1; k cat/K M = (1.2±0.2)×105 M-1 s-1 at pH 5.2). Stopped-flow studies of this BrO2 - decomposition as a function of pH show that 1) the two-electron oxidized heme, compound I (Cpd I), is the primary accumulating heme intermediate during O2 evolution in acidic solution, 2) Cpd I and its one-electron reduction product, compound II (Cpd II) are present in varying ratios at intermediate pHs, and 3) only Cpd II is observed at pH 9.0. The pH dependences of Cpd I and Cpd II populations both yield a pK a of 6.7±0.1 in good agreement with the pK a of DaCld activity with ClO2 -. The observation of a protein-based amino acid radical (AA•) whose appearance coincides with that of Cpd II supports the hypothesis that conversion of Cpd I to Cpd II occurs via proton-coupled electron transfer (PCET) from a heme-pocket amino acid to the oxidized porphyrinate of Cpd I to yield a dead-end decoupled state in which the holes decay at different rates. The site of the amino acid radical is tentatively assigned to Y118, which serves as a H-bond donor to propionate 6 (P6). The favoring of Cpd II:AA• accumulation in alkaline solution is consistent with the amino acid oxidation being rate limited by transfer of its proton to P6 having pK a 6.7. Examination of reaction mixtures comprising DaCld and ClO2 - by resonance Raman and electron paramagnetic resonance spectroscopy reveal formation of Cpd II and •ClO2, which forms in preference to the analogous to AA• in the BrO2 - reaction. Addition of ClO- to Cpd II did not yield O2. Together these results are consistent with heterolytic cleavage of the O-BrO- and O-ClO- bonds yielding Cpd I, which is the catalytically active intermediate. The long-lived Cpd II that forms subsequently, is inactive toward O2 production, and diminishes the amount of enzyme available to cycle through the active Cpd I intermediate.

Insights into the Thermodynamics and Kinetics of Amino-Acid Radicals in Proteins.

Some oxidoreductase enzymes use redox-active tyrosine, tryptophan, cysteine, and/or glycine residues as one-electron, high-potential redox (radical) cofactors. Amino-acid radical cofactors typically perform one of four tasks-they work in concert with a metallocofactor to carry out a multielectron redox process, serve as storage sites for oxidizing equivalents, activate the substrate molecules, or move oxidizing equivalents over long distances. It is challenging to experimentally resolve the thermodynamic and kinetic redox properties of a single-amino-acid residue. The inherently reactive and highly oxidizing properties of amino-acid radicals increase the experimental barriers further still. This review describes a family of stable and well-structured model proteins that was made specifically to study tyrosine and tryptophan oxidation-reduction. The so-called α3X model protein system was combined with very-high-potential protein film voltammetry, transient absorption spectroscopy, and theoretical methods to gain a comprehensive description of the thermodynamic and kinetic properties of protein tyrosine and tryptophan radicals.

Statistical analysis of ENDOR spectra

Electron-nuclear double resonance (ENDOR) measures the hyperfine interaction of magnetic nuclei with paramagnetic centers and is hence a powerful tool for spectroscopic investigations extending from biophysics to material science. Progress in microwave technology and the recent availability of commercial electron paramagnetic resonance (EPR) spectrometers up to an electron Larmor frequency of 263 GHz now open the opportunity for a more quantitative spectral analysis. Using representative spectra of a prototype amino acid radical in a biologically relevant enzyme, the [Formula: see text] in Escherichia coli ribonucleotide reductase, we developed a statistical model for ENDOR data and conducted statistical inference on the spectra including uncertainty estimation and hypothesis testing. Our approach in conjunction with 1H/2H isotopic labeling of [Formula: see text] in the protein unambiguously established new unexpected spectral contributions. Density functional theory (DFT) calculations and ENDOR spectral simulations indicated that these features result from the beta-methylene hyperfine coupling and are caused by a distribution of molecular conformations, likely important for the biological function of this essential radical. The results demonstrate that model-based statistical analysis in combination with state-of-the-art spectroscopy accesses information hitherto beyond standard approaches.

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

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.

Nonexchange Sorption of Amino Acids on an AV-17 Anion Exchanger: Quantum-Chemical Simulation

The energy of interaction between various amino acids (glycine, alanine, and phenylalanine) and an AV-17 strongly basic anion exchanger in chloride and nitrate ionic forms under nonexchange absorption conditions is assessed by quantum chemistry calculations. We found that the amount of water in the starting fragments of sorption participants, the counterion of the anion exchanger, and the amino acid radical play an important role in energy and in the ratio of different intermolecular interactions (Coulomb, hydrophobic, and hydrogen bonds) to form ion-molecular structures in the ion-exchanger phase.

One Electron Multiple Proton Transfer in Model Organic Donor-Acceptor Systems: Implications for High Frequency EPR.

EPR spectroscopy is an important spectroscopic method for identification and characterization of radical species involved in many biological reactions. The tyrosyl radical is one of the most studied amino acid radical intermediates in biology. Often in conjunction with histidine residues, it is involved in many fundamental biological electron and proton transfer processes, such as in the water oxidation in photosystem II. As biological processes are typically extremely complicated and hard to control, molecular bio-mimetic model complexes are often used to clarify the mechanisms of the biological reactions. Here we present theoretical calculations to investigate the sensitivity of magnetic resonance parameters to proton-coupled electron transfer events, as well as conformational substates of the molecular constructs which mimic the tyrosine-histidine (Tyr-His) pairs found in a large variety of proteins. Upon oxidation of the phenol, the Tyr analogue, these complexes can perform not only one-electron one-proton transfer (EPT), but also one-electron two-proton transfers (E2PT). It is shown that in aprotic environment the gX-components of the electronic g-tensor are extremely sensitive to the first proton transfer from the phenoxyl oxygen to the imidazole nitrogen (EPT product), leading to a significant increase of the gX-value of up to 0.003, but are not sensitive to the second proton transfer (E2PT product). In the latter case the change of the gX-value is much smaller (ca. 0.0001), which is too small to be distinguished even by high frequency EPR. The 14N hyperfine values are also too similar to allow differentiation between the different protonation states in EPT and E2PT. The magnetic resonance parameters were also calculated as a function of the rotation angles around single bonds. It was demonstrated that rotation of the phenoxyl group results in large positive changes (>0.001) in the gX-values. Analysis of the data reveals that the main source of these changes is related to the strength of the H-bond between phenoxyl oxygen and the proton(s) on N1 and N2 positions of the imidazole.

Kinetic analysis of amino acid radicals formed in H2O2-driven CuI LPMO reoxidation implicates dominant homolytic reactivity

Lytic polysaccharide monooxygenases (LPMOs) have been proposed to react with both [Formula: see text] and [Formula: see text] as cosubstrates. In this study, the [Formula: see text] reaction with reduced Hypocrea jecorina LPMO9A (CuI-HjLPMO9A) is demonstrated to be 1,000-fold faster than the [Formula: see text] reaction while producing the same oxidized oligosaccharide products. Analysis of the reactivity in the absence of polysaccharide substrate by stopped-flow absorption and rapid freeze-quench (RFQ) electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) yields two intermediates corresponding to neutral tyrosyl and tryptophanyl radicals that are formed along minor reaction pathways. The dominant reaction pathway is characterized by RFQ EPR and kinetic modeling to directly produce CuII-HjLPMO9A and indicates homolytic O-O cleavage. Both optical intermediates exhibit magnetic exchange coupling with the CuII sites reflecting facile electron transfer (ET) pathways, which may be protective against uncoupled turnover or provide an ET pathway to the active site with substrate bound. The reactivities of nonnative organic peroxide cosubstrates effectively exclude the possibility of a ping-pong mechanism.

Impact of Local Electrostatics on the Redox Properties of Tryptophan Radicals in Azurin: Implications for Redox-Active Tryptophans in Proton-Coupled Electron Transfer.

Tyrosine and tryptophan play critical roles in facilitating proton-coupled electron transfer (PCET) processes essential to life. The local protein environment is anticipated to modulate the thermodynamics of amino acid radicals to achieve controlled, unidirectional PCET. Herein, square-wave voltammetry was employed to investigate the electrostatic effects on the redox properties of tryptophan in two variants of the protein azurin. Each variant contains a single redox-active tryptophan, W48 or W108, in a unique and buried protein environment. These tryptophan residues exhibit reversible square-wave voltammograms. A Pourbaix plot, representing the reduction potentials versus pH, is presented for the non-H-bonded W48, which has potentials comparable to those of tryptophan in solution. The reduction potentials of W108 are seen to be increased by more than 100 mV across the same pH range. Molecular dynamics shows that, despite its buried indole ring, the N-H of W108 hydrogen bonds with a water cluster, while W48 is completely excluded from interactions with water or polar groups. These redox properties provide insight into the role of the protein in tuning the reactivity of tryptophan radicals, a requirement for controlled biological PCET.

Fast reaction of carbon free radicals with flavonoids and other aromatic compounds

Many theoretical and experimental studies have shown that the principal initial biological targets of free radicals are nucleic acids, lipids and proteins. The reaction normally generates carbon-centered radicals which can propagate molecular damage either directly or after formation of new reactive species following reaction with oxygen. Overall damage prevention is therefore best achieved by repair of the carbon radicals before they initiate further reactions. Recent studies have shown that the repair cannot be achieved by normal levels of the endogenous antioxidants glutathione, ascorbate or urate. Since their concentrations are well regulated and cannot be enhanced by oral intake, we have investigated the effectiveness of flavonoids and other polyphenols as potential carbon radical repair agents, because their levels in vivo can be significantly enhanced by diet. Pulse radiolysis measurements of the rate constants of repair of amino acid radicals by several polyphenols showed reversible formation of radical-polyphenol adducts 100–1000 times faster than previously reported for the bimolecular stoichiometric reactions of flavonoids i.e. with rate constants in the order of 1010 M−1s−1. Adduct formation depended only on the presence of a carbon-centered radical and an aromatic moiety in the reactants, without the involvement of redox reactions at the phenolic groups. Formation of adducts lowered the reactivity of the radicals. Our results suggest that flavonoids, polyphenols and many of their metabolites can effectively reduce the damaging potential of carbon radicals at concentrations achievable in vivo by diets rich in fruits and vegetables.