Amino Acid Radicals Research Articles

Repair of Protein Radicals by Antioxidants

AbstractIn vivo, proteins are the main targets for radicals and other reactive species. Their reactions result in formation of amino acid radicals on the protein surface that often yield tryptophan and tyrosyl radicals or, in the presence of O2, protein peroxyl radicals and hydroperoxides. All these species may propagate damage to biomolecules. Low molecular weight antioxidants, such as ascorbate, urate, and glutathione, are part of the defense system and function by repairing damaged proteins. We briefly review the existing knowledge about protein and amino acid radicals and their repair by antioxidants, including results of our investigations. The main question addressed is whether the antioxidants ascorbate, urate, and glutathione are able to repair amino acid radicals in model compounds and in proteins in vitro by pulse radiolysis. We show that ascorbate and urate repair tryptophan and tyrosyl radicals efficiently and inhibit proton‐coupled electron transfer from tyrosine residues to tryptophan radicals in a number of proteins. In contrast, repair by glutathione is much slower. Ascorbate also rapidly reduces the peroxyl radicals of the N‐acetylamide derivatives of glycine, alanine, and proline, whereas glutathione reduces peroxyl radicals in lysozyme. In vivo urate, ascorbate, and glutathione may prevent biological damage or, at least, reduce its rate, because they: (a) repair tryptophan and tyrosyl radicals in proteins and (b) reduce protein peroxyl radicals to the corresponding protein hydroperoxides. Most likely, in vivo, ascorbate and glutathione do not inhibit the reaction of C‐centered amino acid radicals with O2. Glutathione is less efficient that urate and ascorbate in repairing protein radicals; furthermore, the resulting glutathiyl radical is harmful. Ascorbate may be the more important repair agent in cells and tissues characterized by high ascorbate concentrations, such as the lens and brain; urate may be mainly responsible for repair in tissue compartments with higher urate concentrations, such as in plasma and saliva.

The kinetics of the reaction of nitrogen dioxide with iron(II)- and iron(III) cytochrome c

The reactions of NO2 with both oxidized and reduced cytochrome c at pH 7.2 and 7.4, respectively, and with N-acetyltyrosine amide and N-acetyltryptophan amide at pH 7.3 were studied by pulse radiolysis at 23°C. NO2 oxidizes N-acetyltyrosine amide and N-acetyltryptophan amide with rate constants of (3.1±0.3)×105 and (1.1±0.1)×106M−1s−1, respectively. With iron(III)cytochrome c, the reaction involves only its amino acids, because no changes in the visible spectrum of cytochrome c are observed. The second-order rate constant is (5.8±0.7)×106M−1s−1 at pH 7.2. NO2 oxidizes iron(II)cytochrome c with a second-order rate constant of (6.6±0.5)×107M−1s−1 at pH 7.4; formation of iron(III)cytochrome c is quantitative. Based on these rate constants, we propose that the reaction with iron(II)cytochrome c proceeds via a mechanism in which 90% of NO2 oxidizes the iron center directly—most probably via reaction at the solvent-accessible heme edge—whereas 10% oxidizes the amino acid residues to the corresponding radicals, which, in turn, oxidize iron(II). Iron(II)cytochrome c is also oxidized by peroxynitrite in the presence of CO2 to iron(III)cytochrome c, with a yield of ~60% relative to peroxynitrite. Our results indicate that, in vivo, NO2 will attack preferentially the reduced form of cytochrome c; protein damage is expected to be marginal, the consequence of formation of amino acid radicals on iron(III)cytochrome c.

Generation of guanine-amino acid cross-links by a free radical combination mechanism.

A direct method has been developed for the in vitro synthesis of stable DNA-protein cross-links (DPC's) between guanine and amino acids (lysine and arginine). This method employs the combination of guanine neutral radicals, G(-H)˙, and side-chain C-centered amino acid radicals. The latter were generated indirectly after first causing the selective photoionization of 2-aminopurine (2AP) embedded in the oligonucleotide, 5'-d(CC[2AP]TCGCTACC), by intense nanosecond 308 nm excimer laser pulses. The 2AP radical cation deprotonates rapidly to form the 2AP(-H)˙ neutral radical which, in turn, oxidizes the nearby guanine to form the neutral guanine G(-H)˙ radical, as described previously (Shafirovich et al., J. Phys. Chem. B, 2001, 105, 8431). In parallel, the hydrated electrons, generated by the photoionization of 2AP, are scavenged by nitrous oxide to generate hydroxyl radicals. In the presence of a large excess of the amino acids, the hydroxyl radicals oxidize the latter to produce C-centered amino acid radicals that combine with the G(-H)˙ radicals to form the guanine-amino acid cross-linked oligonucleotide product. Analogous products were generated by photoionizing the free nucleoside, 2',3',5'-tri-O-acetylguanosine, (tri-O-Ac-Guo), using intense nanosecond 266 nm Nd:YAG laser pulse irradiation. The guanine-amino acid cross-links thus produced site-specifically positioned either in oligonucleotides, or in the free nucleoside tri-O-Ac-Guo were isolated by HPLC methods and identified by high resolution LC-TOF/MS and LC-MS/MS methods. The possibility that analogous guanine-amino acid cross-linked products could be formed in vivo using single hit radical generation mechanisms during oxidative stress is discussed.

Formal Reduction Potential of 3,5-Difluorotyrosine in a Structured Protein: Insight into Multistep Radical Transfer

The reversible Y-O•/Y-OH redox properties of the α3Y model protein allow access to the electrochemical and thermodynamic properties of 3,5-difluorotyrosine. The unnatural amino acid has been incorporated at position 32, the dedicated radical site in α3Y, by in vivo nonsense codon suppression. Incorporation of 3,5-difluorotyrosine gives rise to very minor structural changes in the protein scaffold at pH values below the apparent pK (8.0±0.1) of the unnatural residue. Square-wave voltammetry on α3(3,5)F2Y provides an E°'(Y-O•/Y-OH) of 1026±4 mV versus the normal hydrogen electrode (pH 5.70±0.02) and shows that the fluoro substitutions lower the E°' by -30±3 mV. These results illustrate the utility of combining the optimized α3Y tyrosine radical system with in vivo nonsense codon suppression to obtain the formal reduction potential of an unnatural aromatic residue residing within a well-structured protein. It is further observed that the protein E°' values differ significantly from peak potentials derived from irreversible voltammograms of the corresponding aqueous species. This is notable because solution potentials have been the main thermodynamic data available for amino acid radicals. The findings in this paper are discussed relative to recent mechanistic studies of the multistep radical-transfer process in Escherichia coli ribonucleotide reductase site-specifically labeled with unnatural tyrosine residues.

Development of non-standard arginine residue parameters for use with the AMBER force fields

Amino acid radicals are often involved as intermediates in biological processes, but are difficult to capture by experiment. Computational modeling can be employed to study the features of the species involved. The neutral arginyl radical has previously been detected experimentally using ECD and ETD spectroscopy. Protonation of the radical can occur on the guanidinium carbon, depending on the peptide structure and protein environment. Accurate force fields are essential for reproducing the conformational and dynamic behavior of these intermediates. New AMBER ff99 parameters for the arginyl radical and hydrogenated arginyl side chains are presented based on ab initio quantum chemical calculations.

Function of the diiron cluster of Escherichia coli class Ia ribonucleotide reductase in proton-coupled electron transfer.

The class Ia ribonucleotide reductase (RNR) from Escherichia coli employs a free-radical mechanism, which involves bidirectional translocation of a radical equivalent or "hole" over a distance of ~35 Å from the stable diferric/tyrosyl-radical (Y122(•)) cofactor in the β subunit to cysteine 439 (C439) in the active site of the α subunit. This long-range, intersubunit electron transfer occurs by a multistep "hopping" mechanism via formation of transient amino acid radicals along a specific pathway and is thought to be conformationally gated and coupled to local proton transfers. Whereas constituent amino acids of the hopping pathway have been identified, details of the proton-transfer steps and conformational gating within the β sununit have remained obscure; specific proton couples have been proposed, but no direct evidence has been provided. In the key first step, the reduction of Y122(•) by the first residue in the hopping pathway, a water ligand to Fe1 of the diferric cluster was suggested to donate a proton to yield the neutral Y122. Here we show that forward radical translocation is associated with perturbation of the Mössbauer spectrum of the diferric cluster, especially the quadrupole doublet associated with Fe1. Density functional theory (DFT) calculations verify the consistency of the experimentally observed perturbation with that expected for deprotonation of the Fe1-coordinated water ligand. The results thus provide the first evidence that the diiron cluster of this prototypical class Ia RNR functions not only in its well-known role as generator of the enzyme's essential Y122(•), but also directly in catalysis.

Competition of Charge- versus Radical-Directed Fragmentation of Gas-Phase Protonated Cysteine Sulfinyl Radicals

The fragmentation behavior of various cysteine sulfinyl ions (intact, N-acetylated, and O-methylated), new members of the gas-phase amino acid radical ion family, was investigated by low-energy collision-induced dissociation (CID). The dominant fragmentation channel for the protonated cysteine sulfinyl radicals ((SO•)Cys) was the radical-directed Cα-Cβ homolytic cleavage, resulting in the formation of glycyl radical ions and loss of CH2SO. This channel, however, was not observed for protonated N-acetylated cysteine sulfinyl radicals (Ac-(SO•)Cys); instead, charge-directed H2O loss followed immediately by SH loss prevailed. Counterintuitively, the H2O loss did not derive from the carboxyl group but involved the sulfinyl oxygen, a proton, and a Cβ hydrogen atom. Theoretical calculations suggested that N-acetylation significantly increases the barrier (~14 kcal mol(-1)) for the radical-directed fragmentation channel because of its reduced capability to stabilize the thus-formed glycyl radical ions via the captodative effect. N-Acetylation also assists in moving the proton to the sulfinyl site, which reduces the barrier for H2O loss. Our studies demonstrate that for cysteine sulfinyl radical ions, the stability of the product ions (glycyl radical ions) and the location of the charge (proton) can significantly modulate the competition between radical- and charge-directed fragmentation.

Electron transfer without metals. How amino acid radicals (mis)behave

Tyrosine participates in electron transfer reactions in biology. The reaction mechanism of ribonucleotide reductase involves electron transfers between Tyr, Trp, and Cys. The Tyr radical may also be formed as the result of reaction with HO•. Tyrosine is the ultimate electron donor in a pecking order consisting of Met, Trp, and Tyr ≈ Cys. As the range of published electrode potentials for Trp at pH 7 is +0.56 – +1.05 V, and +0.68 – +0.94 V for Tyr, we set out to determine a consistent set of electrode potentials. By cyclic voltammetry, we measured E°′(TyrO•,H+/TyrOH) = +0.98±0.02 V and E°′(Trp•,H+/TrpH) = +1.00±0.01 V, thus ΔE°′ = 0.02±0.02 V (K = 2) for electron transfer between Trp•and Tyr; by pulse radiolysis, we found K = 9, corresponding to ΔE°′ = 0.05 V. We recommend that the presently accepted values of E°′ for Trp (+1.05 V) and Tyr (+0.94 V), be replaced with +1.01 V and +0.97 V, respectively. For nitrotyrosine, we found +1.11±0.02 V by cyclic voltammetry. Pulse radiolysis experiments described in the literature suggest that the value of E°′(Cys•,H+/CysH) is similar to that of the Tyr couple; Schöneich and coworkers, and we have shown, however, that the S‐centered radical is in rapid equilibrium with C‐centered radicals in Cys and neighbouring amino acids (10.1021/tx3000494 and 10.1021/tx300045f).Supported by the Swiss National Science Foundation and the ETH.

The Effect of Thermal and Ultrasonic Treatment on Amino Acid Composition, Radical Scavenging and Reducing Potential of Hydrolysates Obtained from Simulated Gastrointestinal Digestion of Cowpea Proteins

The effect of thermal and ultrasonic treatment of cowpea proteins (CP) on amino acid composition, radical scavenging and reducing potential of hydrolysates (CPH) obtained from in vitro simulated gastrointestinal digestion of CP was evaluated. Hydrolysis of native and treated CP with gastrointestinal pepsin and pancreatin yielded CPH that displayed antioxidant activities based on oxygen radical scavenging capacity (ORAC), ferric reducing antioxidant power (FRAP) and superoxide radical scavenging activity (SRSA). CPH derived from the treated CP yielded higher ORAC values than CPH from untreated proteins. However, lower significant FRAP and SRSA values were observed for these samples compared to untreated CPH (p < 0.05). Amino acid analysis indicated that CP processing decreased total sulphur-containing amino acids in the hydrolysates, particularly cysteine. The amount of cysteine appeared to be positively related to FRAP and SRSA values of CPH samples, but not ORAC. The results indicated that thermal and ultrasonic processing of CP can reduce the radical scavenging and reducing potential of the enzymatic hydrolysates possibly due to the decreased amounts of cysteine. Since the hydrolysates were generated with gastrointestinal enzymes, it is possible that the resulting compounds are produced to exert some health functions during normal consumption of cowpea.

ChemInform Abstract: Radiation‐Induced Radicals and Radical Ions in Amino Acids and Peptides

AbstractReview: 111 refs.

Efficient depletion of ascorbate by amino acid and protein radicals under oxidative stress

Ascorbate levels decrease in organisms subjected to oxidative stress, but the responsible reactions have not been identified. Our earlier studies have shown that protein C-centered radicals react rapidly with ascorbate. In aerobes, these radicals can react with oxygen to form peroxyl radicals. To estimate the relative probabilities of the reactions of ascorbate with protein C- and O-centered radicals, we measured by pulse radiolysis the rate constants of the reactions of C-centered radicals in Gly, Ala, and Pro with O2 and of the resultant peroxyl radicals with ascorbate. Calculations based on the concentrations of ascorbate and oxygen in human tissues show that the relative probabilities of reactions of the C-centered amino acid radicals with O2 and ascorbate vary between 1:2.6 for the pituitary gland and 1:0.02 for plasma, with intermediate ratios for other tissues. The high frequency of occurrence of Gly, Ala, and Pro in proteins and the similar reaction rate constants of their C-centered radicals with O2 and their peroxo-radicals with ascorbate suggest that our results are also valid for proteins. Thus, the formation of protein C- or O-centered radicals in vivo can account for the loss of ascorbate in organisms under oxidative stress.

Photochemical tyrosine oxidation with a hydrogen-bonded proton acceptor by bidirectional proton-coupled electron transfer

Amino acid radical generation and transport are fundamentally important to numerous essential biological processes to which small molecule models lend valuable mechanistic insights. Pyridyl-amino acid-methyl esters are appended to a rhenium(I) tricarbonyl 1,10-phenanthroline core to yield rhenium-amino acid complexes with tyrosine ([Re]-Y-OH) and phenylalanine ([Re]-F). The emission from the [Re] center is more significantly quenched for [Re]-Y-OH upon addition of base. Time-resolved studies establish that excited-state quenching occurs by a combination of static and dynamic mechanisms. The degree of quenching depends on the strength of the base, consistent with a proton-coupled electron transfer (PCET) quenching mechanism. Comparative studies of [Re]-Y-OH and [Re]-F enable a detailed mechanistic analysis of a bidirectional PCET process.

Multifrequency Electron Paramagnetic Resonance Characterization of PpoA, a CYP450 Fusion Protein that Catalyzes Fatty Acid Dioxygenation

PpoA is a fungal dioxygenase that produces hydroxylated fatty acids involved in the regulation of the life cycle and secondary metabolism of Aspergillus nidulans . It was recently proposed that this novel enzyme employs two different heme domains to catalyze two separate reactions: within a heme peroxidase domain, linoleic acid is oxidized to (8R)-hydroperoxyoctadecadienoic acid [(8R)-HPODE]; in the second reaction step (8R)-HPODE is isomerized within a P450 heme thiolate domain to 5,8-dihydroxyoctadecadienoic acid. In the present study, pulsed EPR methods were applied to find spectroscopic evidence for the reaction mechanism, thought to involve paramagnetic intermediates. We observe EPR resonances of two distinct heme centers with g-values typical for Fe(III) S = (5)/(2) high-spin (HS) and Fe(III) S = (1)/(2) low-spin (LS) hemes. (14)N ENDOR spectroscopy on the S = (5)/(2) signal reveals resonances consistent with an axial histidine ligation. Reaction of PpoA with the substrate leads to the formation of an amino acid radical on the early millisecond time scale concomitant to a substantial reduction of the S = (5)/(2) heme signal. High-frequency EPR (95- and 180-GHz) unambiguously identifies the new radical as a tyrosyl, based on g-values and hyperfine couplings from spectral simulations. The radical displays enhanced T(1)-spin-lattice relaxation due to the proximity of the heme centers. Further, EPR distance measurements revealed that the radical is distributed among the monomeric subunits of the tetrameric enzyme at a distance of approximately 5 nm. The identification of three active paramagnetic centers involved in the reaction of PpoA supports the previously proposed reaction mechanism based on radical chemistry.

Pulsed electron–electron double resonance: beyond nanometre distance measurements on biomacromolecules

PELDOR (or DEER; pulsed electron-electron double resonance) is an EPR (electron paramagnetic resonance) method that measures via the dipolar electron-electron coupling distances in the nanometre range, currently 1.5-8 nm, with high precision and reliability. Depending on the quality of the data, the error can be as small as 0.1 nm. Beyond mere mean distances, PELDOR yields distance distributions, which provide access to conformational distributions and dynamics. It can also be used to count the number of monomers in a complex and allows determination of the orientations of spin centres with respect to each other. If, in addition to the dipolar through-space coupling, a through-bond exchange coupling mechanism contributes to the overall coupling both mechanisms can be separated and quantified. Over the last 10 years PELDOR has emerged as a powerful new biophysical method without size restriction to the biomolecule to be studied, and has been applied to a large variety of nucleic acids as well as proteins and protein complexes in solution or within membranes. Small nitroxide spin labels, paramagnetic metal ions, amino acid radicals or intrinsic clusters and cofactor radicals have been used as spin centres.

EPR parameters of amino acid radicals in P. eryngii versatile peroxidase and its W164Y variant computed at the QM/MM level

Quantum mechanics/molecular mechanics (QM/MM) methods, employing density functional theory (DFT), have been used to compute the electron paramagnetic resonance (EPR) parameters of tryptophan and tyrosyl radical intermediates involved in the catalytic cycle of Pleurotus eryngii versatile peroxidase (VP) and its W164Y variant, respectively. These radicals have been previously experimentally detected and characterized both in the two-electron and one-electron activated forms of the enzymes. In this work, the well-studied W164 radical in VP has been chosen for calibration purposes because its spectroscopic properties have been extensively studied by multifrequency EPR and ENDOR spectroscopies. Using a B3LYP/CHARMM procedure, appropriately accounting for electrostatic, such as hydrogen bonding, and steric environmental interactions, a good agreement between the calculated and measured EPR parameters for both radicals has been achieved; g-tensors, hyperfine coupling constants (hfcc) and Mulliken spin densities have been correlated to changes in geometries, hydrogen bond networks and electrostatic environment, with the aim of understanding the influence of the protein surroundings on EPR properties. In addition, the present calculations demonstrate, for VP, the formation of a neutral tryptophan radical, hydrogen bonded to the nearby E243, via a stepwise electron and proton transfer with earlier involvement of a short-lived tryptophan cationic species. Instead, for W164Y, the QM/MM dynamics simulation shows that the tyrosine oxidation proceeds via a concerted electron and proton transfer and is accompanied by a significant reorganization of residues and water molecules surrounding the tyrosyl radical.

Catalytic Mechanism of a Heme and Tyrosyl Radical-Containing Fatty Acid α-(Di)oxygenase

The steady-state catalytic mechanism of a fatty acid α-(di)oxygenase is examined, revealing that a persistent tyrosyl radical (Tyr379(•)) effects O(2) insertion into C(α)-H bonds of fatty acids. The initiating C(α)-H homolysis step is characterized by apparent rate constants and deuterium kinetic isotope effects (KIEs) that increase hyperbolically upon raising the concentration of O(2). These results are consistent with H(•) tunneling, transitioning from a reversible to an irreversible regime. The limiting deuterium KIEs increase from ∼30 to 120 as the fatty acid chain is shortened from that of the native substrate. In addition, activation barriers increase in a manner that reflects decreased fatty acid binding affinities. Anaerobic isotope exchange experiments provide compelling evidence that Tyr379(•) initiates catalysis by H(•) abstraction. C(α)-H homolysis is kinetically driven by O(2) trapping of the α-carbon radical and reduction of a putative peroxyl radical intermediate to a 2(R)-hydroperoxide product. These findings add to a body of work which establishes large-scale hydrogen tunneling in proteins. This particular example is novel because it involves a protein-derived amino acid radical.

Study of the structure of free radicals in gamma: irradiated amino acid derivatives

Electron paramagnetic resonance spectroscopy has been used for the study of the free radicals produced in the powders of L-arginine monohydrochloride, ala-ala and DL-β-leucine gamma-irradiated at room temperature. The structures of the radicals formed in compounds were determined as NH2(NH)NHCHCH2CH2CH(NH2)COOH, CH3CHCOOH and (CH3)2C CH(NH2)CH2COOH respectively. The paramagnetic species were found to be stable at room temperature more than three months. It was determined that unpaired electron interacted with nearby protons and 14N nucleus in these radicals. The g values of the radicals and the hyperfine structure constants of the free electron were also measured. These results were compared with the earlier studies in amino acid and derivatives radicals and discussed.

Long-lived radicals of amino acids induced by X-ray radiation are the source of hydrogen peroxide in aqueous medium

The formation of long-lived radicals in the solutions of casein and its hydrolysate with an equimolar mixture of amino acids was compared by measuring the X-ray-induced chemiluminescence. It was shown that free amino acids constituting the protein produce long-lived radicals. It was demonstrated that some amino acids (Leu, Ile, Val, Ser, Trp, Met, Pro, Arg, Gly, Phe) emit light of visible spectrum over a long period of time after the irradiation, which indicates the generation of long-lived radicals of these amino acids. The half-life times of these radicals are several hours. Dissolving irradiated dry amino acids capable of luminescing over a long time gives rise to the formation of hydrogen peroxide in aqueous medium.

Studies of heterogeneous interactions between N-acetyl- N′-methyl- l-α-amino acid amides and urea molecules in water at 298.15 K

The enthalpies of solution of N-acetyl- N′-methyl- l-α-threoninamide, N-acetyl- N′-methyl- l-α-tyrosinamide, N-acetyl- N′-methyl- l-α-histidinamide, N-acetyl- N′-methyl- l-α-tryptophanamide have been measured in water and in aqueous urea solutions at 298.15 K. The mixing enthalpies of aqueous urea solution with aqueous N-acetyl- N′-methyl- l-α-valinamide solution and their respective enthalpies of dilution by water have been determined at 298.15 K. The experimental results were used to obtain the enthalpic coefficients of the interaction between amino acid amides and urea molecules in water based on McMillan–Mayer's model. The values of the interaction parameters were interpreted in terms of the hydrophobic or hydrophilic properties of the amino acid radicals in the examined amide molecules and the influence on their interactions with a urea molecule in water.

Electron Capture Dissociation Product Ion Abundances at the X Amino Acid in RAAAA-X-AAAAK Peptides Correlate with Amino Acid Polarity and Radical Stability

We present mechanistic studies aimed at improving the understanding of the product ion formation rules in electron capture dissociation (ECD) of peptides and proteins in Fourier transform ion cyclotron resonance mass spectrometry. In particular, we attempted to quantify the recently reported general correlation of ECD product ion abundance (PIA) with amino acid hydrophobicity. The results obtained on a series of model H-RAAAAXAAAAK-OH peptides confirm a direct correlation of ECD PIA with X amino acid hydrophobicity and polarity. The correlation factor (R) exceeds 0.9 for 12 amino acids (Ile, Val, His, Asn, Asp, Glu, Gln, Ser, Thr, Gly, Cys, and Ala). The deviation of ECD PIA for seven outliers (Pro is not taken into consideration) is explained by their specific radical stabilization properties (Phe, Trp, Tyr, Met, and Leu) and amino acid basicity (Lys, Arg). Phosphorylation of Ser, Thr, and Tyr decreases the efficiency of ECD around phosphorylated residues, as expected. The systematic arrangement of amino acids reported here indicates a possible route toward development of a predictive model for quantitative electron capture/transfer dissociation tandem mass spectrometry, with possible applications in proteomics.