Spectroscopic properties of tyrosyl radicals in dipeptides

Redox-active tyrosine residues play important roles in long-distance electron reactions in enzymes, including prostaglandin H synthase, galactose oxidase, ribonucleotide reductase, and photosystem II. Magnetic resonance and vibrational spectroscopy provide methods with which to study the structures of redox-active amino acids in proteins. In this report, ultraviolet photolysis was used to generate tyrosyl radicals from polycrystalline tyrosinate or dipeptides, and the structure of the radical was investigated with EPR and reaction-induced FT-IR spectroscopy at 77 K. Photolysis at 77 K is expected to generate a neutral tyrosyl radical through oxidation of the aromatic ring. EPR and FT-IR results obtained from (13)C-labeled tyrosine were consistent with that expectation. Surprisingly, labeling of the tyrosyl amino group with (15)N also resulted in isotope-shifted bands in the photolysis spectrum. The force constant of a NH deformation mode increased when the tyrosyl radical was generated. These data suggest an interaction between the pi system of the tyrosyl radical and the amino group. In spectra acquired from the dipeptides, evidence for a sequence-dependent interaction between the tyrosyl radical and the amide bond of the dipeptide was also obtained. We postulate that perturbation of the amino or the amide/imide groups may occur through a spin polarization mechanism, which is indirectly detected as a change in NH force constant. This conclusion is supported by density functional calculations, which suggest a conformationally sensitive delocalization of spin density onto the amino and carboxylate groups of the tyrosyl radical. These experiments provide a step toward a detailed spectral interpretation for protein-based tyrosyl radicals.     

PRIMARY PRODUCTS IN THE FLASH PHOTOLYSIS OF TRYPTOPHAN*

There has been considerable interest in the photochemistry of tryptophan in connection with ultraviolet inactivation of enzymes. Earlier flash photolysis work has demonstrated that the hydrated electron (e^-_aq) is an initial product in the irradiation of indole derivatives, accompanied by a longer-lived transient absorption near 500 nm attributed to an aromatic radical species[1–5]. Similar transients were observed in a recent flash photolysis study of lysozyme[6] in which it was proposed that inactivation is a consequence of electron ejection from 1 to 2 essential tryptophan residues in the active center. However, there has been uncertainty concerning the tryptophan radical structure and its relationship to the triplet state and radical spectra reported for tryptophan photolysis in low-temperature rigid media. This note reports a flash photolysis investigation of L-tryptophan (Trp) and 1-Methyl-L-tryptophan (1-MeTrp) undertaken to clarify these points. The flash photolysis apparatus and methods employed are described in Ref. [6].     

Photochemical inactivation of trypsin

Abstract —The quantum yield for inactivation of aqueous trypsin fits the expression φ_f=Σ_rf_rφ‘_r, where f_r, is the fraction of incident light absorbed by residues of type r and the φ’_r are constants. The values φ‘_trp= 0.012, φ_tyr= 0.005 and φ’_eys= 0.10, obtained at pH 3 in the wavelength range 240–290 nm, are attributed to independent events by comparing with quantum yields of the initial photochemical products and permanent residue destruction. The proposed inactivating processes are photoionization of one essential tryptophyl residue, photolysis of one essential cystyl residue, and splitting of an essential cystyl residue induced by light absorption in a nearby tyrosyl residue. The initial photochemical process from pH 3–7 identified by flash photolysis is the ejection of electrons from approximately two tryptophyl residues, leading to the formation of the disulfide bridge electron adduct and the hydrated electron. It is proposed that one photoionized tryptophyl residue is permanently disrupted and the other is restored through a back reaction that leads to a damaged, active enzyme form. An enhanced inactivation quantum yield at flash photolysis light intensities is attributed to a biphotonic process. A model based on one-photon photoionization of tryptophan from a short-lived precursor of the fluorescent state and the biphotonic photoionization of tryptophan via the triplet state is consistent with the experimental results.     

INTERMEDIATES IN THE ROOM TEMPERATURE FLASH PHOTOLYSIS OF ADENINE AND SOME OF ITS DERIVATIVES

The transient absorption spectra of aqueous solutions of adenine, 2′-deoxyadenosine, 2′-deoxyadenosine-5′-phosphate and 2′-deoxyadenylyl-(3′-5′)-2′-deoxyadenosine have been determinated at different pH values using conventional flash photolysis. Reactives intermediates produced in the flash photolysis of these adenine derivatives present similar absorption regions: two higher intensity bands in the UV and 560–720 nm wavelength region and a third weaker band at 420–560 nm. On the basis of the effects produced by triplet quenchers and/or electron scavengers the bands have been assigned to hydrated electrons, radical cations, radical anions and/or neutral radicals resulting from neutralization reactions of the charged radicals. The results indicate that the bases photoionize via a triplet state under these conditions.     

A FLASH PHOTOLYSIS STUDY OF 5-METHOXYINDOLE

Abstract— The microsecond flash photolysis of 5-methoxyindole in aqueous solutions has been studied at γ_exc≥ 290 nm. Transients identified in this time realm in neutral solutions are: e_aq^-, the 5-methoxyindole radical cation (γ_max≅ 440 nm), the neutral transient with γ_max≅ 530 nm) and an unidentified oxygen sensitive transient with γ_max≅ 435 nm. Radical cations and e^-_aq are shown to be produced in equal amounts consistent with a photoionization process as the only source of both transients. H⁺ quenching of fluorescence and radical cation production gives equivalent Stern-Volmer constants indicating that photoionization occurs from the fluorescent state. The unidentified oxygen sensitive transient exhibits a pK _a of2–2.5 and is quenched at lower pH values indicating that it also has a fluorescent state precursor.     

Functional molecules from single wall carbon nanotubes. Photoinduced solubility of short single wall carbon nanotube residues by covalent anchoring of 2,4,6-triarylpyrylium units

Raw, micrometric HiPCO single wall carbon nanotube (SWNT) material was submitted to harsh acid oxidative treatment with a 3:1 H2SO4/HNO3 mixture to give short residues of SWNT (s-SWNT, <200 nm length measured by TEM). s-SWNT was functionalized through the tip carboxylic groups by peptide bonds using 3-mercatopropanamine linkers that subsequently were reacted with 2,6-diphenyl-4-(4-vinylbiphenyl)pyrylium using azobis(isobutyronitrile) as a radical initiator. After purification by dialysis, the resulting s-SWNT having covalently linked through an ethylthiopropylamide tether the strong electron-transfer pyrylium photosensitizer (Py-sSWNT) was characterized by solution 1H NMR spectroscopy (observation of specific signals due to the heterocyclic protons). Emission spectroscopy shows that the fluorescence of 2,6-diphenyl-4-(4-dodecylthiobiphenyl)pyrylium (Py-SC12) tetrafluoroborate (a model compound to the tethered pyrylium moiety in Py-sSWNT) (lambdaem 533 nm) is quenched by s-SWNT and vice versa that the emission of s-SWNT (lambdaem 330 nm) is quenched by Py-SC12. Depending on the excitation wavelength, Py-sSWNT exhibits dual emission corresponding to each of the two moieties, but with much less intensity than each of the model components independently. Laser flash photolysis of model Py-SC12 allows detection of the triplet (lambdaT-T 750 nm, tau 11.7 micros) and the much longer-lived pyrylium centered radical (lambdamax 525 nm, tau 147 mus). The latter species arises from photoinduced electron transfer from the sulfur atom, as the donor, to the pyrylium heterocycle in its electronic excited-state, as the electron acceptor. Laser flash photolysis (355 nm) of Py-sSWNT also allows detection of the pyrylium centered radical together with a broad absorption spanning from 200 to 500 nm and peaking at 280 nm. The latter band is absent in the laser flash photolysis of the model s-SWNT and was attributed to the electron hole localized on the nanotube moiety of Py-SWNT. The most remarkable effect of the steady-state irradiation is a 1 order of magnitude increase in the solubility of Py-sSWNT. According to TEM images this photoinduced solubility can be attributed to the debundling of the nanotubes due to photoinduced charge separation through the nanotube walls. In addition to exemplify how molecular compounds with photoresponsive properties can be derived from SWNT materials, the observation of photoinduced solubility can serve to develop SWNT layers suitable for photolithography patterning.     

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.     

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.     

Flash photolysis and pulse radiolysis studies on collagen Type I in acetic acid solution

An investigation of the photochemical properties of collagen Type I in acetic acid solution was carried out using nanosecond laser irradiation. The transient spectra of collagen solution excited at 266 nm show two bands. One of them with maximum at 295 nm and the second one with maximum at 400 nm. The peak at 400 nm is assigned to tyrosyl radicals. The first peak of the transient absorption spectra at 295 nm is probably due to photoionisation producing collagen radical cation. The transient for collagen solution in acetic acid at 640 nm was not observed. It is evidence that there is no hydrated electron in the irradiated collagen solution. The reactions of hydrated electrons and (*)OH radicals with collagen have been studied by pulse radiolysis. In the absorption spectra of products resulting from the reaction of collagen with e(aq)(-) no characteristic maximum absorption in UV and visible light region has been observed. In the absorption spectra of products resulting from the reaction of the hydroxyl radicals with collagen two bands have been observed. The first one at 320 nm and the second one at 405 nm. Reaction of (*)OH radicals with tyrosine residues in collagen chains gives rise to Tyr phenoxyl radicals (absorption at 400 nm).     

THE BIPHOTONIC PHOTOIONIZATION OF CHLORPROMAZINE DURING CONVENTIONAL FLASH PHOTOLYSIS: SPIN TRAPPING RESULTS WITH 5,5-DIMETHYL-1-PYRROLINE-N-OXIDE

A novel combination of conventional flash photolysis and electron spin resonance (ESR) spin-trapping has been used to demonstrate that photoionization of chlorpromazine (CPZ), and the concomitant production of hydrated electron, occurs through a stepwise biphotonic mechanism during conventional flash photolysis at wavelengths above 290 nm. The production of hydrated electron in the flash photolysis experiment has been monitored and quantified through the use of the spin trapping agent, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The effects of nitrous oxide, varying concentrations of CPZ and DMPO, and a range of flash intensities on the ESR spectra of the observed spin adducts of DMPO are discussed. The use of ESR spin trapping to monitor hydrated electron yields in flash photolysis experiments has the potential to permit the use of a much wider range of flash intensities than is typically possible with conventional optical experiments. Thus, there is a greater possibility of distinguishing between monophotonic and biphotonic processes.     

THE PHOTOREDUCTION OF FLAVINS BY AMINO ACIDS AND EDTA. A CONTINUOUS AND FLASH PHOTOLYSIS STUDY

Abstract— Primary and secondary photochemical processes in oxygen-free aqueous solution have been characterised for FMN alone and in the presence of EDTA and four amino acids using nanosecond and microsecond flash photolysis and continuous photolysis techniques. The relative contributions of oneelectron and two-electron (group or hydride transfer) reactions to the deactivation of the triplet has been determined by comparing the radical concentration (560 nm) with the bleaching of the ground state (446 nm). It was concluded that one-electron reactions (hydrogen atom or electron abstraction) are the major mode of reactivity of the flavin triplet state with all the suhstrates studied.
The nature of the reactions of the flavin semiquinone radical have been studied quantitatively by microsecond flash photolysis. These secondary reactions consist of either a 'back reaction' between the flavin and substrate radicals (tryptophan or glycyl-tyrosine) or the transfer of a second electron (or hydrogen atom) from the substrate radical to the flavin radical (EDTA, methionine and possibly cysteine) to form reduced flavin and oxidised substrate. From a comparison of the quantum yields of formation of reduced flavin using 'flash' and continuous irradiation, an additional pathway for the decay of the flavin radical is suggested to occur at low light intensities in the presence of glycyl-tyrosine or histidine.     

Gaining an insight into the photoreactivity of a drug in a protein environment: a case study on nalidixic acid and serum albumin

The binding of nalidixic acid (NA) with human and bovine serum albumin (HSA and BSA) in buffer solution at pH 7.4 was investigated using circular dichroism (CD), UV absorption and fluorescence spectroscopy. Global analysis of multiwavelength spectroscopic data afforded the equilibrium constants of the most stable noncovalent drug/protein adducts of 1:1 and 2:1 stoichiometry and their individual CD, UV absorption, and fluorescence spectra. The primary binding site of the drug was located in subdomain IIIA (Sudlow Site II), whereas the secondary one was assigned to subdomain IIA. Conformational and CD calculations afforded the binding geometries. In the complexes, the fluorescence of the protein was strongly quenched by energy transfer and that of the drug was suppressed by electron transfer. Laser flash photolysis at 355 nm evidenced the formation of a radical pair consisting of a tyroxyl radical (lambdamax = 410 nm) and a reduced nalidixate anion radical NA(2-)* (lambdamax = 640 nm) with quantum yield of 0.4-0.5. Strong evidence was obtained that the process that involves Tyr411 in HSA (Tyr409 in BSA). A further transient with lambdamax approximately 780 nm observed in HSA was attributed to oxidation of the -(S200-S246)- bridge upon electron transfer to NA(-)*. Decay of the confined radical pairs occurred with rates approximately 10(7) s(-1). Formation of covalent drug-protein adducts in mixtures irradiated at lambdairr> 324 nm was proved using HPLC with fluorescence detection.     

A mechanistic and kinetic study of the formation of metal nanoparticles by using synthetic tyrosine-based oligopeptides

Synthetic oligopeptides containing redox-active tyrosine residues have been employed to prepare gold and silver nanoparticles. In this reduction process an electron from the tyrosinate ion of the peptide is transferred to the metal ion at basic pH through the formation of a tyrosyl radical, which is eventually converted to its dityrosine form during the reaction. This reaction mechanism was confirmed from UV-visible, fluorescence, and EPR spectroscopy and was found to be pH-dependent. Transmission electron microscopy measurement shows that the average size and the monodispersity of gold nanoparticles increase as the number of tyrosine residues in the peptide increases. The kinetic study, based on spectrophotometric measurements of the surface plasmon resonance optical property, shows that the rate of formation of gold nanoparticles was much faster at higher pH than at lower pH and was also dependent on the number of tyrosine residues present in the peptide. The dityrosine form of the peptide was found to retain reducing properties like those of tyrosine in basic medium.     

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.     

TRIPLET EXCITED STATE OF COUMARIN AND 4'5' DIHYDROPSORALEN: REACTION WITH NUCLEIC ACID BASES AND AMINO ACIDS

Abstract—The triplet-triplet absorption spectra of coumarin, 5.7 dimethoxycoumarin and the furocoumarin 4'5' dihydropsoralen. a model for 4'5' psoralen-pyrimidine mono adducts, have been determined by the techniques of pulse radiolysis and laser flash photolysis. The extinction coefficients of the triplet transitions have been measured and used to determine the singlet → triplet intersystem crossing quantum yields for 347 nm excitation in water. Reaction rate constants for coumarin and 4'5' dihydropsoralen triplets with various pyrimidine and purine nucleic acid bases, and amino acids, have been measured. Long-lived transient absorptions detected after quenching coumarin and 4'5' dihydropsoralen triplets with tryptophan are assigned to mixtures of the corresponding coumarin radical anion and the tryptophan radical cation. The spectra of the radical anions of coumarin and 4'5' dihydropsoralen were established using pulse radiolysis of the coumarins in aqueous formate. It is suggested that coumarins and furocoumarin triplets are quenched by nucleic acid bases and amino acids via a chargetransfer mechanism.     

Photochemistry and photopolymerization activity of novel perester derivatives of fluorenone: a conventional and laser flash photolysis study

The photochemistry of three novel t-butylperester derivatives of fluorenone was examined and compared with unsubstituted fluorenone and a mono-t-butylperester of benzophenone using both conventional microsecond and nanosecond laser flash photolysis. On conventional microsecond flash photolysis in 2-propanol, all four fluorenone compounds gave transient absorption in the region 300–400 nm due to a ketyl radical formed from the abstraction of a hydrogen atom from the solvent by the upper excited triplet n—π* state of the fluorenone chromophore. This assignment was confirmed by a pH-dependent study on the transient absorption spectra. The nitro-t-butylperester derivative of fluorenone gave additional absorption above 400 nm due to species associated with the nitro group. No evidence for benzoyloxy radical formation could be found in non-hydrogen-atom-donating solvents with microsecond flash photolysis which is associated with homolysis of the perester groups. On nanosecond laser flash photolysis of the fluorenone compounds at 355 nm excitation in acetonitrile and hexa-fluorobenzene, transient absorptions were observed in the region 320–640 nm due to the corresponding triplet states. All the t-butylperester derivatives showed residual absorbances at longer time delays which were tentatively assigned to the corresponding benzoyloxy radicals produced by homolysis of the perester groups. In contrast, the mono-t-butylperester of benzophenone, included for comparison only, showed very weak transient absorption in the region 320–640 nm compared with that of the strong triplet of benzophenone under the same excitation conditions. The triplet absorptions and lifetimes of the fluorenone compounds were correlated with their photopolymerization activities in bulk methylmethacrylate monomer. In oxygenated solutions, the triplet absorptions of fluorenone and benzophenone were effectively quenched; however, long-lived transient growths were observed for all the t-butylperester derivatives. The intensities of these novel transient absorptions appear to correlate with the total number of t-butylperester groups in the fluorenone molecule and tentative assignments are discussed.     

PHOTOLYSIS MECHANISM OF AQUEOUS TYROSINE AND TYROSYL PEPTIDES*

Abstract— Laser flash photolysis at 265 nm has been employed for measuring the initial hydrated electron (e^-_aq) and p-alanylphenoxyl radical (Tyr) in aqueous Tyr, small Tyr peptides and R Nase A. The results indicate that monophotonic photolysis not involving the fluorescent or triplet states is the principal initial process. Equivalent yields of e and Tyr were found in all cases except Tyr, where the Tyr yield was 60% higher than e^-_aq attributed to splitting of the phenolic bond. Computer analysis of e^-_aq and Tyr decays for Tyr indicates the importance of electron-radical recombination in competition with electron scavenging and bimolecular radical-radical reactions. Evidence for intramolecular electron migration has been obtained in cystinyl-bis-Tyr.     

香豆素酮类化合物的瞬态研究——Ⅲ.香豆素酮和碘鎓盐间的电子转移

本工作采用激光闪光光解法对香豆素酮类化合物敏化碘鎓盐化合物问题进行了研究。结果表明:碘鎓盐化合物能通过电子转移机理猝灭香豆素酮的激发三重态。工作中还用甲基紫精(PQ²⁺)为模型化物,观察到它也能使香豆素酮的三重态猝灭,同时可看到在位于610nm处的PQ⁺生成。这些结果说明,在发生电子转移的反应中香豆素酮是电子给体,按Weller公式的计算结果也表明它们之间可发生电子转移反应。     

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.     

Photophysical, photochemical and photobiological properties of pyrrolocoumarins; a new class of photoactive compounds

With the aim of finding new photoactive compounds that may reduce the side effects of 8-methoxypsoralen photochemotherapy we report on some photophysical, photochemical and photobiological properties of recently synthesized pyrrolocoumarins, in particular 4-methyl-N-ethyl-pyrrolo[3,2-g]coumarin (PCNEt) which has an absorption maximum in the UV-A (320-400 nm). Laser (347 nm) flash photolysis studies showed triplet transients that were quenched by O2 and by ground state PCNEt. Singlet minus triplet spectra were broad (350-550 nm) and, at 700 nm, indicated solvated electron and radical production. PCNEt complexes with DNA in the dark and photobinds to thymine but does not form DNA cross-links. PCNEt was phototoxic in yeast with an action spectrum similar to its absorption spectrum. PCNEt showed photohaemolytic activity but was not phototoxic on guinea pig skin. These data suggest that PCNEt may photosensitize via several mechanisms: direct DNA photobinding, photodynamic action, or photoproduction of radicals.