Redox-active tyrosine residues in pentapeptides

Tyrosyl radicals are important in long-range electron transfer in several enzymes, but the protein environmental factors that control midpoint potential and electron-transfer rate are not well understood. To develop a more detailed understanding of the effect of protein sequence on their photophysical properties, we have studied the spectroscopic properties of tyrosyl radicals at 85 K. Tyrosyl radical was generated by UV-photolysis of pentapeptides in polycrystalline samples. The sequence of the pentapeptides was chosen to mimic peptide sequences found in redox-active tyrosine containing enzymes, ribonucleotide reductase and photosystem II. From EPR studies, we report that the EPR line shape of the tyrosyl radical depends on peptide sequence. We also present the first evidence for a component of the tyrosyl radical EPR signal, which decays on the seconds time scale at 85 K. We suggest that this transient results from a spontaneous, small conformational rearrangement in the radical. From FT-IR studies, we show that amide I vibrational bands (1680-1620 cm(-1)) and peptide bond skeletal vibrations (1230-1090 cm(-1)) are observed in the photolysis spectra of tyrosine-containing pentapeptides. From these data, we conclude that oxidation of the tyrosine aromatic ring perturbs the electronic structure of the peptide bond in tyrosine-containing oligopeptides. We also report sequence-dependent alterations in these bands. These results support the previous suggestion (J. Am. Chem. Soc. 2002, 124, 5496) that spin delocalization can occur from the tyrosine aromatic ring into the peptide bond. We hypothesize that these sequence-dependent effects are mediated either by electrostatics or by changes in conformer preference in the peptides. Our findings suggest that primary structure influences the functional properties of redox-active tyrosines in enzymes.     

Normal modes of redox-active tyrosine: conformation dependence and comparison to experiment

Redox-active tyrosine residues play important roles in long-distance electron reactions in enzymes such as prostaglandin H synthase, ribonucleotide reductase, and photosystem II (PSII). Spectroscopic characterization of tyrosyl radicals in these systems provides a powerful experimental probe into the role of the enzyme in mediation of long-range electron transfer processes. Interpretation of such data, however, relies critically on first establishing a spectroscopic fingerprint of isotopically labeled tyrosinate and tyrosyl radicals in nonenzymatic environments. In this report, FT-IR results obtained from tyrosinate, tyrosyl radical (produced by ultraviolet photolysis of polycrystalline tyrosinate), and their isotopologues at 77 K are presented. Assignment of peaks and isotope shifts is aided by density-functional B3LYP/6-311++G(3df,2p)//B3LYP/6-31++G(d,p) calculations of tyrosine and tyrosyl radical in several different charge and protonation states. In addition, characterization of the potential energy surfaces of tyrosinate and tyrosyl radical as a function of the backbone and ring torsion angles provides detailed insight into the sensitivity of the vibrational frequencies to conformational changes. These results provide a detailed spectroscopic interpretation, which will elucidate the structures of redox-active tyrosine residues in complex protein environments. Specific application of these data is made to enzymatic systems.     

ESEEM studies of peptide nitrogen hyperfine coupling in tyrosyl radicals and model peptides

Tyrosyl radicals are important in long-range electron transfer in several enzymes, but the protein environmental factors that control midpoint potential and electron transfer rate are not well understood. To develop a more detailed understanding of the effect of protein sequence, we have performed 14N and 15N electron spin echo envelope modulation (ESEEM) measurements on tyrosyl radical, generated either in polycrystalline tyrosinate or in its 15N-labeled isotopomer, by UV photolysis. 14N-ESEEM was also performed on tyrosyl radical generated in tyrosine-containing pentapeptide samples. Simulation of the 14N- and 15N-tyrosyl radical ESEEM measurements yielded no significant isotropic hyperfine splitting to the amine or amide nitrogen; the amplitude of the anisotropic, nitrogen hyperfine coupling (0.21 MHz) was consistent with a dipole-dipole distance of 3.0 A. Density functional theory was used to calculate the isotropic and anisotropic hyperfine couplings to the amino nitrogen in four different tyrosyl radical conformers. Comparison with the simulated data suggested that the lowest energy radical conformer, generated in tyrosine at pH 11, has a 76 degrees Calpha-Cbeta-C1'-C2' ring and a -73 degrees C-Calpha-Cbeta-C1' backbone dihedral angle. In addition, the magnitude, orientation, and asymmetry of the nuclear quadrupole coupling tensor were derived from analysis of the tyrosyl radical 14N-ESEEM. The simulations showed differences in the coupling and orientation of the nuclear quadrupole tensor, when the tyrosinate and pentapeptide samples were compared. These results suggest sequence- or conformation-induced changes in the ionic character of the NH bond in different tyrosine-containing peptides.     

Photolysis of poly(amino acid)s at 77K: An ESR study

The mechanism of the ultraviolet (λ > 250 nm) degradation of poly(amino acid)s has been studied by ESR spectroscopy at 77 K. In the aliphatic poly(amino acid)s of glycine, alanine and valine, absorption of energy occurs predominantly in the peptide group, and the initial degradation reaction is scission of the CONH bond. The imino radical, ·NHCHR, abstracts the main chain H from the CHR group, or the tertiary H from valine, to give secondary carbon radicals. The acyl radical, CHRCO·, readily loses CO to form a chain-end radical, CHR·. The aromatic poly(amino acid)s of phenylalanine and tyrosine absorb energy mainly in the phenyl chromophore and bond scission occurs in the side chain. The mechanism of photolysis of the poly(amino acid)s differs from that of the N-acetyl amino acids (except for tyrosine) due to the presence of the labile carboxyl group.     

Multifrequency high-field EPR study of the tryptophanyl and tyrosyl radical intermediates in wild-type and the W191G mutant of cytochrome c peroxidase.

Multifrequency (95, 190, and 285 GHz) high-field electron paramagnetic resonance (EPR) spectroscopy has been used to characterize radical intermediates in wild-type and Trp191Gly mutant cytochrome c peroxidase (CcP). The high-field EPR spectra of the exchange-coupled oxoferryl--trytophanyl radical pair that constitutes the CcP compound I intermediate [(Fe(IV)=O) Trp*(+)] were analyzed using a spin Hamiltonian that incorporated a general anisotropic spin-spin interaction term. Perturbation expressions of this Hamiltonian were derived, and their limitations under high-field conditions are discussed. Using numerical solutions of the completely anisotropic Hamiltonian, its was possible to simulate accurately the experimental data from 9 to 285 GHz using a single set of spin parameters. The results are also consistent with previous 9 GHz single-crystal studies. The inherent superior resolution of high-field EPR spectroscopy permitted the unequivocal detection of a transient tyrosyl radical that was formed 60 s after the addition of 1 equiv of hydrogen peroxide to the wild-type CcP at 0 degrees C and disappeared after 1 h. High-field EPR was also used to characterize the radical intermediate that was generated by hydrogen peroxide addition to the W191G CcP mutant. The g- values of this radical (g(x)= 2.00660, g(y) = 2.00425, and g(z)= 2.00208), as well as the wild-type transient tyrosyl radical, are essentially identical to those obtained from the high-field EPR spectra of the tyrosyl radical generated by gamma-irradiation of crystals of tyrosine hydrochloride (g(x)= 2.00658, g(y) = 2.00404, and g(z) = 2.00208). The low g(x)-value indicated that all three of the tyrosyl radicals were in electropositive environments. The broadening of the g(x) portion of the HF-EPR spectrum further indicated that the electrostatic environment was distributed. On the basis of these observations, possible sites for the tyrosyl radical(s) are discussed.     

Redox-active tyrosine residues: role for the peptide bond in electron transfer

Photosystem II (PSII) catalyzes the light-driven oxidation of water and reduction of plastoquinone. In PSII, redox-active tyrosine Z conducts electrons between the primary chlorophyll donor and the manganese cluster, which is the catalytic site. In this report, difference FT-IR spectroscopy is used to show that oxidation of redox-active tyrosine Z causes perturbations of the peptide bond. PSII data were acquired on control samples, as well as samples in which tyrosine was 2H4 (ring)-labeled. Comparison to model compound data, acquired both from tyrosinate and its 2H4 isotopomer, was performed. The PSII FT-IR spectrum exhibited vibrational bands that are assignable to imide and amide vibrational modes. In previous work, we have shown that oxidation of tyrosinate perturbs the terminal amino group of tyrosinate (Ayala, I.; Range, K.; York, D.; Barry, B. A. J. Am. Chem. Soc. 2002, 124, 5496-5505). Density functional calculations on tyrosinate supported the interpretation that the perturbation is due to spin delocalization onto the amino group. In tyrosine-containing dipeptides, perturbations of the peptide bond were observed. Therefore, the imide and amide perturbations observed here are attributed to spin delocalization into the peptide bond in PSII. Migration of the electron hole in PSII may be consistent with peptide bond involvement in tyrosyl radical-based electron-transfer reactions.     

SPIN-TRAPPING AND ESR STUDIES OF THE DIRECT PHOTOLYSIS OF AROMATIC AMINO ACIDS, DIPEPTIDES, TRIPEPTIDES AND POLYPEPTIDES IN AQUEOUS SOLUTIONS—I. PHENYLALANINE AND RELATED COMPOUNDS

Abstract— The direct UV photolysis of l -Phe and peptides containing l -Phe in aqueous solutions has been investigated at room temperature. The short-lived free radicals formed during photolysis were spin-trapped by t -nitrosobutane and identified by electron spin resonance. During the photolysis of l -Phe the decarboxylation and the deamination radicals were spin-trapped. For N-formyl and N-acetyl- l -Phe the decarboxylation radicals were observed. For dipeptides containing Phe the decarboxylation radicals were observed and in some cases the deamination radicals from the N-terminal residue were found. For the tripeptides Gly- l -Phe- l -Ala and Gly-Gly- l -Phe, the C-terminal decarboxylation radical was spin trapped; for l -Phe-Gly-Gly only the deamination radical of the N-terminal residue could be detected. However, for Gly- l -Phe-Gly, five different radicals were identified. The results of the spin-trapping experiments of the 260 nm photolysis of RNase-S-peptide, containing 20 amino acid residues, was interpreted in terms of a chain scission between the alpha carbon of the Phe residue and the adjacent carbonyl group.     

Modeling the active site of cytochrome oxidase: synthesis and characterization of a cross-linked histidine-phenol

A cross-linked histidine-phenol compound was synthesized as a chemical analogue of the active site of cytochrome c oxidase. The structure of the cross-linked compound (compound 1) was verified by IR, (1)H and (13)C NMR, mass spectrometry, and single-crystal X-ray analysis. Spectrophotometric titrations indicated that the pK(a) of the phenolic proton on compound 1 (8.34) was lower than the pK(a) of tyrosine (10.1) or of p-cresol (10.2). This decrease in pK(a) is consistent with the hypothesis that a cross-linked histidine-tyrosine may facilitate proton delivery to the binuclear site in cytochrome c oxidase. Time-resolved optical absorption spectra of compound 1 at room temperature, generated by excitation at 266 nm in the presence and absence of dioxygen, indicated a species with absorption maxima at approximately 330 and approximately 500 nm, which we assign to the phenoxyl radical of compound 1. The electron paramagnetic resonance (EPR) spectra of compound 1, obtained after UV photolysis, confirmed the generation of a paramagnetic species at low temperature. Because the cross-linked compound lacks beta-methylene protons, the EPR line shape was dramatically altered when compared to that of the tyrosyl radical. However, simulation of the EPR line shape and measurement of the isotropic g value was consistent with a small coupling to the imidazole nitrogen and with little spin density perturbation in the phenoxyl ring. The ground-state Fourier transform infrared (FT-IR) spectrum of compound 1 showed that addition of the imidazole ring perturbs the frequency of the tyrosine ring stretching vibrations. The difference FT-IR spectrum, associated with the oxidation of the cross-linked compound, detected significant perturbations of the phenoxyl radical vibrational bands. We postulate that phenol oxidation produces a small delocalization of spin density onto the imidazole nitrogen of compound 1, which may explain its unique optical spectral properties.     

Structure of the Paramagnetic Products of Thiocyanate Photolysis at 77 K

Solid paramagnetic products of thiocyanate photolysis were studied at 77 K by EPR and diffuse reflectance spectroscopy. Ab initio calculations of the electronic structure of these products have been carried out. Direct UV irradiation can generate the SCN⁰ radical due to ionization of the thiocyanate ion. Interaction of the SCN⁰ radical with the neighboring thiocyanate ion forms another radical product of photolysis — (SCN)₂ ^- radical ion.     

Two tyrosyl radicals stabilize high oxidation states in cytochrome C oxidase for efficient energy conservation and proton translocation

The reaction of oxidized bovine cytochrome c oxidase (bCcO) with hydrogen peroxide (H(2)O(2)) was studied by electron paramagnetic resonance (EPR) to determine the properties of radical intermediates. Two distinct radicals with widths of 12 and 46 G are directly observed by X-band EPR in the reaction of bCcO with H(2)O(2) at pH 6 and pH 8. High-frequency EPR (D-band) provides assignments to tyrosine for both radicals based on well-resolved g-tensors. The wide radical (46 G) exhibits g-values similar to a radical generated on L-Tyr by UV-irradiation and to tyrosyl radicals identified in many other enzyme systems. In contrast, the g-values of the narrow radical (12 G) deviate from L-Tyr in a trend akin to the radicals on tyrosines with substitutions at the ortho position. X-band EPR demonstrates that the two tyrosyl radicals differ in the orientation of their β-methylene protons. The 12 G wide radical has minimal hyperfine structure and can be fit using parameters unique to the post-translationally modified Y244 in bCcO. The 46 G wide radical has extensive hyperfine structure and can be fit with parameters consistent with Y129. The results are supported by mixed quantum mechanics and molecular mechanics calculations. In addition to providing spectroscopic evidence of a radical formed on the post-translationally modified tyrosine in CcO, this study resolves the much debated controversy of whether the wide radical seen at low pH in the bovine enzyme is a tyrosine or tryptophan. The possible role of radical formation and migration in proton translocation is discussed.     

Isolation of a Neutral Boron‐Containing Radical Stabilized by a Cyclic (Alkyl)(Amino)Carbene

Utilizing a cyclic (alkyl)(amino)carbene (CAAC) as a ligand, neutral CAAC‐stabilized radicals containing a boryl functionality could be prepared by reduction of the corresponding haloborane adducts. The radical species with a duryl substituent was fully characterized by single‐crystal X‐ray structural analysis, EPR spectroscopy, and DFT calculations. Compared to known neutral boryl radicals, the isolated radical species showed larger spin density on the boron atom. Furthermore, the compound that was isolated is extraordinarily stable to high temperatures under inert conditions, both in solution and in the solid state. Electrochemical investigations of the radical suggest the possibility to generate a stable formal boryl anion species.     

A dual sensor spin trap for use with EPR spectroscopy

Redox active metal ions, carbon-centered radicals, and oxygen-centered radicals are important to oxidative stress. A radical detector combining a nitrone spin trap, a phenol, and a cyclopropane radical clocklike unit was prepared and used with EPR spectroscopy to detect and distinguish between hydroxyl radicals, methyl radicals, and iron(III) ions. Iron(III) reacts with the phenol unit inducing opening of the cyclopropane ring and cyclization to generate a stable nitroxyl radical.     

Nanosecond generation of tyrosyl radicals via laser-initiated decaging of oxalate-modified amino acids

We describe a general method for the unimolecular photochemical generation of tyrosyl radicals from a diaryl oxalate ester platform on the nanosecond time scale. Symmetric and asymmetric tyrosine oxalate esters have been prepared in gram quantities. Direct photocleavage of the oxalate linkage by laser flash photolysis affords tyrosyl radicals within 50 ns. This approach provides unnatural caged amino acids that may be incorporated into model and biological systems for the study of proton-coupled electron transfer in enzymatic catalysis.     

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.     

EPR spectral and laser photolysis study of radical pairs and their annihilation in quinone-doped pyrocatechol monocrystals

Conclusions The possibility of studying radical pairs in monocrystals by laser photolysis has been demonstrated. Comparison of the optical absorption spectra and EPR signals indicated that the radical pairs in 3,6-di-tert-butylpyrocatechol monocrystals with added 3,6-di-tertbutyl-o-benzoquinone at 77 K are comprised from the corresponding semiquinone radicals.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 5, pp. 1166–1167, May, 1988.     

Radical trapping properties of 3-aryl-2H-benzo[1,4]oxazin-4-oxides

A series of 3-aryl-2H-benzo[1,4]oxazin-4-oxides was prepared, and their ability to trap free radicals was investigated by EPR spectroscopy. In organic solvents, these compounds were able to efficiently scavenge all carbon- and oxygen-centered radicals tested, giving very persistent aminoxyls, except with superoxide anion whose spin adducts were unstable. The main feature of these nitrones as spin traps lies in the possibility to recognize the initial radical trapped. In fact, besides a g-factor and aminoxyl nitrogen EPR coupling constant dependence on the species trapped, the EPR spectra also show different patterns due to hyperfine splittings characteristic of the radical scavenged. This last important feature was investigated by means of density functional theory calculations.     

FLASH PHOTOLYSIS OF RIBONUCLEASE A

Abstract— Flash photolysis spectra show that ultraviolet irradiation of RNase (Λ > 250 nm) at pH 11.5 generates the hydrated electron and a long-lived transient with absorption maxima at 390 nm and 410 nm, attributed to the phenoxyl type radical from tyrosyl residues. Comparison of the initial yields with flash photolysis spectra obtained from aqueous tyrosine and mixtures of the chromophoric amino acids indicates that 3–4 tyrosyl residues are photoionized in the primary act. This process is almost completely quenched at pH 1–9, even though the p -alanylphenoxyl radical is obtained with tyrosine over this pH range and the accompanying electron is observed at pH 7. The negative result is not altered by denaturation of RNase with 8 M urea or heating to 70°C, suggesting that a primary chain interaction is responsible for the suppression of tyrosyl residue photolysis. This mechanism is supported by flash photolysis spectra of small peptides, showing that the initial radical yield from tyrosylglycylglycine is strongly quenched compared to tyrosine when the phenolic group is protonated. Comparion of this work with published results on fluorescence and inactivation quantum yields indicates that photochemical electron ejection from RNase in alkaline solutions takes place in the dissociable residues and does not contribute to loss of enzymic activity.     

Electron-electron double resonance in electron spin echo: Model biradical systems and the sensitized photolysis of decalin

Model systems, comprising frozen glassy solutions of stabilized radicals and biradicals of the nitroxyl type, have been used to test the applicability of electron-electron double resonance in electron spin echo (ELDOR ESE) in studies of the spatial distributions of free radicals arranged in groups in solids. The method was used to investigate the spatial distribution of alkyl radicals generated by the sensitized photolysis of glassy naphthalene solutions in decalin at 77 K. and detected radical pairs.     

Photochemically generated carotenoid radicals on Nafion film and silica gel: an EPR and ENDOR study

Carotenoid cation radicals have been observed at 120 K, by EPR and proton ENDOR measurements, to be formed upon 77 K photolysis of thin films of Nafion or silica gel coated with the carotenoids, β-carotene and canthaxanthin. The powder ENDOR spectra consist of resolvable lines due to couplings larger than 2.8 MHz but smaller than 17 MHz assigned to the methyl protons of the carotenoid cation radical and to an -proton of the planar polyene chain.     

Proton-coupled electron transfer in a biomimetic peptide as a model of enzyme regulatory mechanisms

Proton-coupled electron-transfer reactions are central to enzymatic mechanism in many proteins. In several enzymes, essential electron-transfer reactions involve oxidation and reduction of tyrosine side chains. For these redox-active tyrosines, proton transfer couples with electron transfer, because the phenolic pKA of the tyrosine is altered by changes in the tyrosine redox state. To develop an experimentally tractable peptide system in which the effect of proton and electron coupling can be investigated, we have designed a novel amino acid sequence that contains one tyrosine residue. The tyrosine can be oxidized by ultraviolet photolysis or electrochemical methods and has a potential cross-strand interaction with a histidine residue. NMR spectroscopy shows that the peptide forms a beta-hairpin with several interstrand dipolar contacts between the histidine and tyrosine side chains. The effect of the cross-strand interaction was probed by electron paramagnetic resonance and electrochemistry. The data are consistent with an increase in histidine pKA when the tyrosine is oxidized; the effect of this thermodynamic coupling is to increase tyrosyl radical yield at low pH. The coupling mechanism is attributed to an interstrand pi-cation interaction, which stabilizes the tyrosyl radical. A similar interaction between histidine and tyrosine in enzymes provides a regulatory mechanism for enzymatic electron-transfer reactions.