Resonance Raman Studies Research Articles

Resonance Raman study on indoleamine 2,3-dioxygenase: Control of reactivity by substrate-binding

Resonance Raman spectra of ligand-bound complexes including the 4-phenylimidazole complex and of free and L-Trp-bound forms of indoleamine 2, 3-dioxygenase in the ferric state were examined. Effects on the vinyl and propionate substituent groups of the heme were detected in a ligand-dependent fashion. The effects of phenyl group of 4-phenylimidazole on the vinyl and propionate Raman bands were evident when compared with the case of imidazole ligand. Substrate binding to the ferrous protein caused an upshift of the iron–histidine stretching mode by 3cm−1, indicating an increase in negativity of the imidazole ring, which favors the O–O bond cleavage. The substrate binding event is likely to be communicated from the heme distal side to the iron–histidine bond through heme substituent groups and the hydrogen-bond network which includes water molecules, as identified in an X-ray structure of a 4-phenylimidazole complex. The results provide evidence for fine-tuning of the reactivity of O–O bond cleavage by the oxygenated heme upon binding of L-Trp.

Structure and Reactivity of Thiazolium Azo Dyes: UV–Visible, Resonance Raman, NMR, and Computational Studies of the Reaction Mechanism in Alkaline Solution

UV-visible absorption, resonance Raman, and (1)H NMR spectroscopy, allied with density functional theory (DFT) calculations, have been used to study the structure, bonding, and alkaline hydrolysis mechanism of the cationic thiazloium azo dye, 2-[2-[4-(diethylamino)phenyl]diazenyl]-3-methyl-thiazolium (1a), along with a series of six related dyes with different 4-dialkylamino groups and/or other phenyl ring substituents (2a-c, 3a-c) and the related isothiazolium azo dye, 5-[2-[4-(dimethylamino)phenyl]diazenyl]-2-methyl-isothiazolium (4). These diazahemicyanine dyes are calculated to have a similar low-energy structure that is cis, trans at the (iso)thiazolium-azo group, and for which the calculated Raman spectra provide a good match with the experimental data; the calculations on these structures are used to assign and discuss the transitions giving rise to the experimental spectra, and to consider the bonding and its variation between the dyes. UV-visible, Raman, and NMR spectra recorded from minutes to several weeks after raising the pH of an aqueous solution of 1a to ca. 11.5 show that the dominant initial step in the reaction is loss of diethylamine to produce a quinonimine (ca. hours), with subsequent reactions occurring on longer time scales (ca. days to weeks); kinetic analyses give a rate constant of 2.6 × 10(-2) dm(3) mol(-1) s(-1) for reaction of 1a with OH(-). UV-visible spectra recorded on raising the pH of the other dyes in solution show similar changes that are attributed to the same general reaction mechanism, but with different rate constants for which the dependence on structure is discussed.

Effect of chlorine substitution on triplet state structure of thioxanthone: A time‐resolved resonance Raman study

Substitution plays an important role in determining the triplet state reactivity. In this paper, we have studied the effect of chlorine substitution on the triplet state structure and the reactivity of thioxanthone (TX). We have employed time‐resolved resonance Raman technique to understand the structure of the lowest triplet excited state of 2‐chlorothioxanthone (CTX). The experimental findings have been corroborated with the computational results using density functional theory. Akin to the parent compound (TX), coexistence of two lowest triplet states has been observed in case of CTX, which has been substantiated using resonant probe wavelength dependence study. The relative contribution of 3n–π* to 3π–π* to the equilibrated triplet state has been found to be more for CTX compared to TX suggesting increase in the triplet state reactivity after the substitution. The above observation has been further supported by the flash photolysis experiments. Copyright © 2013 John Wiley & Sons, Ltd.

Nonheme oxoiron(iv) complexes of pentadentate N5 ligands: spectroscopy, electrochemistry, and oxidative reactivity

Oxoiron(IV) species have been found to act as the oxidants in the catalytic cycles of several mononuclear nonheme iron enzymes that activate dioxygen. To gain insight into the factors that govern the oxidative reactivity of such complexes, a series of five synthetic S = 1 [Fe(IV)(O)(L(N5))](2+) complexes has been characterized with respect to their spectroscopic and electrochemical properties as well as their relative abilities to carry out oxo transfer and hydrogen atom abstraction. The Fe=O units in these five complexes are supported by neutral pentadentate ligands having a combination of pyridine and tertiary amine donors but with different ligand frameworks. Characterization of the five complexes by X-ray absorption spectroscopy reveals Fe=O bonds of ca. 1.65 Å in length that give rise to the intense 1s→3d pre-edge features indicative of iron centers with substantial deviation from centrosymmetry. Resonance Raman studies show that the five complexes exhibit ν(Fe=O) modes at 825-841 cm(-1). Spectropotentiometric experiments in acetonitrile with 0.1 M water reveal that the supporting pentadentate ligands modulate the E(1/2)(IV/III) redox potentials with values ranging from 0.83 to 1.23 V vs. Fc, providing the first electrochemical determination of the E(1/2)(IV/III) redox potentials for a series of oxoiron(IV) complexes. The 0.4-V difference in potential may arise from differences in the relative number of pyridine and tertiary amine donors on the L(N5) ligand and in the orientations of the pyridine donors relative to the Fe=O bond that are enforced by the ligand architecture. The rates of oxo-atom transfer (OAT) to thioanisole correlate linearly with the increase in the redox potentials, reflecting the relative electrophilicities of the oxoiron(IV) units. However this linear relationship does not extend to the rates of hydrogen-atom transfer (HAT) from 1,3-cyclohexadiene (CHD), 9,10-dihydroanthracene (DHA), and benzyl alcohol, suggesting that the HAT reactions are not governed by thermodynamics alone. This study represents the first investigation to compare the electrochemical and oxidative properties of a series of S = 1 Fe(IV)=O complexes with different ligand frameworks and sheds some light on the complexities of the reactivity of the oxoiron(IV) unit.

On the electronic spectroscopy of the iso-polyhalomethanes

The iso-polyhalomethanes are important reactive intermediates, displaying intense near-UV absorption bands that have been assigned to the S0→S3 transition on the basis of Time-Dependent Density Functional Theory (TDDFT) calculations. In this work, theory and multi-dimensional Franck–Condon (FC) analysis are used to model the electronic spectra of selected iso-polyhalomethanes. The S0→S3 transition approximately corresponds to a π–π∗ transition on the halocarbocation subunit, which induces significant geometry changes. The calculated multimode FC profiles capture features of the experimental spectra of the matrix-isolated species, and are compared with the results of previous Resonance Raman studies of the isomers in solution.

Simultaneous Detection of Two Triplets: A Time-Resolved Resonance Raman Study

Solvents are known to affect the triplet state structure and reactivity. In this paper, we have employed time-resolved resonance Raman (TR3) spectroscopy to understand solvent-induced subtle structural changes in the lowest excited triplet state of thioxanthone. Density functional theory (DFT) combined with the self-consistent reaction field (SCRF) implicit solvation model has been used to calculate the vibrational frequencies in the solvents. Here, we report a unique observation of the coexistence of two triplets, which has been substantiated by the probe wavelength-dependent Raman experiments. The coexistence of two triplets has been further supported by photoreduction experiments carried out at various temperatures.

Redox-Induced Conformational Switching in Photosystem-II-Inspired Biomimetic Peptides: A UV Resonance Raman Study

Long-distance electron transfer (ET) plays a critical role in solar energy conversion, DNA synthesis, and mitochondrial respiration. Tyrosine (Y) side chains can function as intermediates in these reactions. The oxidized form of tyrosine deprotonates to form a neutral tyrosyl radical, Y(•), a powerful oxidant. In photosystem II (PSII) and ribonucleotide reductase, redox-active tyrosines are involved in the proton-coupled electron transfer (PCET) reactions, which are key in catalysis. In these proteins, redox-linked structural dynamics may play a role in controlling the radical's extraordinary oxidizing power. To define these dynamics in a structurally tractable system, we have constructed biomimetic peptide maquettes, which are inspired by PSII. UV resonance Raman studies were conducted of ET and PCET reactions in these β-hairpins, which contain a single tyrosine residue. At pH 11, UV photolysis induces ET from the deprotonated phenolate side chain to solvent. At pH 8.5, interstrand proton transfer to a π-stacked histidine accompanies the Y oxidation reaction. The UV resonance Raman difference spectrum, associated with Y oxidation, was obtained from the peptide maquettes in D(2)O buffers. The difference spectra exhibited bands at 1441 and 1472 cm(-1), which are assigned to the amide II' (CN) vibration of the β-hairpin. This amide II' spectral change was attributed to substantial alterations in amide hydrogen bonding, which are coupled with the Y/Y(•) redox reaction and are reversible. These experiments show that ET and PCET reactions can create new minima in the protein conformational landscape. This work suggests that charge-coupled conformational changes can occur in complex proteins that contain redox-active tyrosines. These redox-linked dynamics could play an important role in control of PCET in biological oxygen evolution, respiration, and DNA synthesis.

UV resonance Raman study of TrpZip2 and related peptides: π-π interactions of tryptophan.

Aromatic interactions are important stabilizing forces in proteins but are difficult to detect in the absence of high-resolution structures. Ultraviolet resonance Raman spectroscopy is used to probe the vibrational signatures of aromatic interactions in TrpZip2, a synthetic β-hairpin peptide that is stabilized by edge-to-face and face-to-face tryptophan π-π interactions. The vibrational markers of isolated edge-to-face π-π interactions are investigated in the related β-hairpin peptide W2W11. The bands that comprise the Fermi doublet exhibit systematic shifts in position and intensity for TrpZip2 and W2W11 relative to the model peptide, W2W9, which does not form aromatic interactions. Additionally, hypochromism of the Bb absorption band of tryptophan in TrpZip2 leads to a decrease in the relative Raman cross-sections of Bb-coupled Raman bands. These results reveal spectral markers for stabilizing tryptophan π-π interactions and indicate that ultraviolet resonance Raman may be an important tool for the characterization of these biological forces.

Site-specific Protein Dynamics in Communication Pathway from Sensor to Signaling Domain of Oxygen Sensor Protein, HemAT-Bs: TIME-RESOLVED ULTRAVIOLET RESONANCE RAMAN STUDY

HemAT-Bs is a heme-based signal transducer protein responsible for aerotaxis. Time-resolved ultraviolet resonance Raman (UVRR) studies of wild-type and Y70F mutant of the full-length HemAT-Bs and the truncated sensor domain were performed to determine the site-specific protein dynamics following carbon monoxide (CO) photodissociation. The UVRR spectra indicated two phases of intensity changes for Trp, Tyr, and Phe bands of both full-length and sensor domain proteins. The W16 and W3 Raman bands of Trp, the F8a band of Phe, and the Y8a band of Tyr increased in intensity at hundreds of nanoseconds after CO photodissociation, and this was followed by recovery in ∼50 μs. These changes were assigned to Trp-132 (G-helix), Tyr-70 (B-helix), and Phe-69 (B-helix) and/or Phe-137 (G-helix), suggesting that the change in the heme structure drives the displacement of B- and G-helices. The UVRR difference spectra of the sensor domain displayed a positive peak for amide I in hundreds of nanoseconds after photolysis, which was followed by recovery in ∼50 μs. This difference band was absent in the spectra of the full-length protein, suggesting that the isolated sensor domain undergoes conformational changes of the protein backbone upon CO photolysis and that the changes are restrained by the signaling domain. The time-resolved difference spectrum at 200 μs exhibited a pattern similar to that of the static (reduced - CO) difference spectrum, although the peak intensities were much weaker. Thus, the rearrangements of the protein moiety toward the equilibrium ligand-free structure occur in a time range of hundreds of microseconds.

Ultrafast Pump–Probe Study of the Excited-State Charge-Transfer Dynamics in Blue Copper Rusticyanin

We have used femtosecond pump-probe spectroscopy to investigate the excited-state dynamics of the anticancer blue copper protein rusticyanin, by exciting its ligand to metal charge-transfer band with 25 fs pump pulses centered at 585 nm. The charge-transfer excited state decays exponentially to the ground state with a time constant of about 230 fs, and its recovery is modulated by coherent oscillations. The Fourier transform of the oscillatory component of the signal provides most of the vibrational modes obtained by means of conventional resonance Raman studies, in addition to the low frequency modes below 80 cm(-1) believed to reflect collective motions of biological relevance.

Oxoferryl-Porphyrin Radical Catalytic Intermediate in Cytochrome bd Oxidases Protects Cells from Formation of Reactive Oxygen Species

The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b(558) that donates electrons to a binuclear heme b(595)/heme d center. The reaction with O(2) and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O(2), the O-O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b(595). Compound I accumulates to 0.75-0.85 per enzyme in agreement with its much higher rate of formation (~20,000 s(-1)) compared with its rate of decay (~1,900 s(-1)). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b(558) before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O-O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O-O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species.

Nuclear resonance vibrational spectra of five-coordinate imidazole-ligated iron(II) porphyrinates.

Nuclear resonance vibrational spectra have been obtained for six five-coordinate imidazole-ligated iron(II) porphyrinates, [Fe(Por)(L)] (Por = tetraphenylporphyrinate, octaethylporphyrinate, tetratolylporphyrinate, or protoporphyrinate IX and L = 2-methylimidazole or 1,2-dimethylimidazole). Measurements have been made on both powder and oriented crystal samples. The spectra are dominated by strong signals around 200-300 cm(-1). Although the in-plane and out-of-plane vibrations are seriously overlapped, oriented crystal spectra allow their deconvolution. Thus, oriented crystal experimental data, along with density functional theory (DFT) calculations, enable the assignment of key vibrations in the spectra. Molecular dynamics are also discussed. The nature of the Fe-N(Im) vibrations has been elaborated further than was possible from resonance Raman studies. Our study suggests that the Fe motions are coupled with the porphyrin core and peripheral groups motions. Both peripheral groups and their conformations have significant influence on the vibrational spectra (position and shape).

UV resonance Raman studies of the NaClO4 dependence of poly-L-lysine conformation and hydrogen exchange kinetics.

We used 204 nm excitation UV Resonance Raman (UVRR) spectroscopy to examine the effects of NaClO(4) on the conformation of poly-L-lysine (PLL). The presence of NaClO(4) induces the formation of α-helix, π-helix/bulge, and turn conformations. The dependence of the AmIII(3) frequency on the peptide Ψ Ramachandran angle allows us to experimentally determine the conformational population distributions and the energy landscape of PLL along the Ramachandran Ψ angle. We also used UVRR to measure the NaClO(4) concentration dependence of PLL amide hydrogen exchange kinetics. Exchange rates were determined by fitting the D(2)O exchanging PLL UVRR AmII' band time evolution. Hydrogen exchange is slowed at high NaClO(4) concentrations. The PLL AmII' band exchange kinetics at 0.0, 0.2, and 0.35 M NaClO(4) can be fit by single exponentials, but the AmII' band kinetics of PLL at 0.8 M NaClO(4) requires a double exponential fit. The exchange rates for the extended conformations were monitored by measuring the C(α)-H band kinetics. These kinetics are identical to those of the AmII' band until 0.8 M NaClO(4) whereupon the extended conformation exchange becomes clearly faster than that of the α-helix-like conformations. Our results indicate that ClO(4)(-) binds to the PLL backbone to protect it from OH(-) exchange catalysis. In addition, ClO(4)(-) binding also slows the conformational exchange between the extended and α-helix-like conformations, probably by increasing the activation barriers for conformational interchanges.

Mesoporous Silica Functionalized by Nickel-Cyclam Molecules: Preparation and Resonance Raman Study

Abstract Mesoporous silica SBA-15 functionalized by (1,4,8,11-tetraazacyclotetradecane) cyclam groups containing nickel ions (Ni-cyclam) was synthesized by two different approaches, and investigated by resonance Raman spectroscopy. Vibrational features of organometallic moleculess are analyzed for (Ni-cyclam) groups grafted in the silica pores. An assignment of bands in resonance Raman spectra was done to monitor the structure and properties of the mesoporous silica material with regard to the methods of synthesis used in this study. It was shown, that Raman scattering can be useful for probing of functionalization's efficiency of mesoporous silica. On the base of the resonance investigation: Raman and EPR spectroscopy, distribution of the functional groups inside pores can be determined. In the present article the Raman spectroscopy is treat as a complementary research to EPR investigation. It was shown that a clustering of the active groups alter significantly the resonance Raman spectra through broadening and shifts of the corresponding bands in comparison with separated molecules. Results obtained from the analysis of the resonance Raman spectra indicate significant differences between the samples prepared by the two procedures. The discussion of the Raman results was referred to EPR results, and on the base of this authors concluded about correct achievement of the functionalization.

Perturbing the Central Hydrophobic Cluster (CHC) of Aβ(10-35) by Incorporation of Fluorinated Phenylalanine Derivatives

One of the putative causes of Alzheimer's disease involves aggregation of misfolded amyloid β (Aβ), a 39-42 residue polypeptide chain, and its subsequent deposition as amyloid plaques. The aggregation process proceeds via a nucleated polymerization mechanism where disordered peptide monomers interact with each other through hydrophobic interactions and rapidly extend and aggregate to eventually form larger fibrils with a highly ordered cross-strand β-sheet structure. It has also been suggested that the aromatic amino acid residues, tyrosine Y10 and phenylalanines (F19 and F20) in the central hydrophobic cluster (CHC) of the peptide play an important role in fibril assembly. In particular, F19 and F20 are suspected to be the drivers of the aggregation mechanism because of their hydrophobicity and aromaticity. In this context perturbation of the CHC through the introduction of non-natural (fluorinated) amino acids is expected to affect the aggregation process. Fluorinated amino acids in particular demonstrate distinct properties dictated by the presence of highly electronegative and hydrophobic fluorine atoms. However such fluorination is known to potentially eliminate the favorable interaction of aromatic hydrogens with the π-electron cloud, which can affect protein-protein interactions. In the present study the introduction of a pentafluoro-Phe in the hydrophobic core of the 26 residue Aβ peptide (Aβ10-35) and its effect on fibril formation has been investigated using circular dichroism (CD) and fluorescence methods which indicate a sequential conformational transition of the peptide from random coil → antiparallel β-sheets → parallel β-sheets. Transition time points have been obtained from these methods and compared to those obtained for the non-fluorinated peptide. UV resonance Raman (UVRR) studies have been performed to probe and characterize the vibrational modes of the fluoro-phenylalanines in the peptide and to explore their effect on the Phe-Phe π-stacking interactions.

Resonance Raman studies of excited state structural displacements of conjugated polymers in donor/acceptor charge transfer complexes

Resonance Raman spectra of poly(2-methoxy-5-(3'-7'-dimethyloctyloxy)-1,4-phenylenevinylene) (MDMO-PPV) and small molecule acceptor blend charge transfer (CT) complexes reveal long and detailed progressions of overtone and combination bands. These features are sensitive to the specific MDMO-PPV/acceptor interactions and enable quantitative calculations of vibrational mode specific displacements of the polymer CT complex.

Ultraviolet Resonance Raman Study of Lipid Mediated Peptide Folding

The interactions between proteins and biological membranes play an important role in many aspects of biochemistry. Thus, the ability to monitor the structural dynamics of membrane proteins is of great interest. In general, hydrophobic peptides are disordered and tend to aggregate in aqueous environments. For example, the amyloid-β (Aβ) peptide, a major component of the insoluble plaques associated with Alzheimer’s disease, is intrinsically disordered under physiological conditions. However, Aβ adopts α-helical structure in membrane mimicking environments. This is not surprising as the hydrophobic region derives from the transmembrane (TM) region of the amyloid precursor protein. More interesting is the fact that low concentrations of organic solvents or surfactants promote aggregation and formation of β-sheet structure. The ability to simultaneously monitor lipid association and study its effect on the secondary structure of amyloidogenic proteins would be of great interest. Recent studies have shown a significantly enhanced amide I mode in the deep-UV resonance Raman (dUVRR) spectra of transmembrane proteins is a marker for lipid association. Positively charged hydrophobic peptides, including the hydrophobic Aβ(25-40) fragment of Aβ, spontaneously insert into anionic lipid bilayers. The application of dUVRR spectroscopy to monitor lipid-association, insertion and folding of these peptides will be presented.

Resonance Raman and EPR spectroscopy studies of untreated spring wheat leaves

Leaves are the structures in plants that have evolved primarily to absorb sunlight and to convert this energy into carbohydrates, which is the essence of the photosynthesis process. In the current study Raman and electron paramagnetic resonance (EPR) spectroscopic methods were applied to investigate the drought stress occurring in the fresh wheat leaves immediately after cutting them off from the rest of the plant. Resonance Raman microspectroscopy mapping allowed us to visualize the changes in concentrations of carotenoids, playing an important role in the photosynthetic membrane, as well as quinones and carbohydrates in the selected areas in plant tissues. EPR spectroscopy was used to detect free radicals in the leaf tissue directly. Besides these very reactive, short lifetime species (monodehydroascorbate–ascorbate radical (MDA)), the long-living semiquinone and carbohydrate radicals have been also detected.

Raman Doping Profiles of Polyelectrolyte SWNTs in Solution

We present a resonance Raman study of electrochemical charge transfer doping on polyelectrolyte single-walled carbon nanotubes (SWNTs) in solution. Changes in the intensity of the radial breathing modes of well-identified SWNTs are measured as a function of the electrochemical potential. The intensity is maximum when the nanotubes are neutral. Unexpectedly, the Raman signal decreases as soon as charges are transferred to the nanotubes, leading to intensity profiles that are triangular for metallic and trapezoidal for semiconducting nanotubes. A key result is that the width in energy of the plateaus for the semiconducting nanotubes is roughly equal to the optical gap (rather than the free carrier gap). While these experiments can be used to estimate the energy levels of individual nanotubes, strong dynamical screening appears to dominate in individual SWNT polyelectrolytes so that only screened energy levels are being probed.

Influence of the chloro substituent position on the triplet reactivity of benzophenone derivatives: a time‐resolved resonance Raman and density functional theory study

A nanosecond time‐resolved resonance Raman (ns‐TR3) spectroscopic investigation of the photoreduction reactions and ability of several chloro‐substituted benzophenone (Cl‐BP) triplets is described. The TR3 results show that the 3‐chlorobenzophenone (3‐Cl‐BP), 4‐chlorobenzophenone (4‐Cl‐BP) and 4,4′‐dichlorobenzophenone (4,4′‐dichloro‐BP) triplets exhibit similar hydrogen abstraction ability with the parent BP triplet. In 2‐propanol, the 3‐Cl‐, 4‐Cl‐ and 4,4′‐dichloro‐diphenylketyl (DPK) radicals were observed and they appear to react with dimethylketyl radicals at the para‐position to form a light absorption transient species. These transient species were characterized with TR3 spectra, and identified with the help of results from density functional theory calculations. In an acetontitrile/water (MeCN:H2O) 1:1 mixed solvent, these DPK radicals were also observed but with slower formation rates. However, the 2‐Cl‐DPK radical was observed to form with a lower yield and a significantly slower formation rate than the other chloro‐substituted benzophenones examined here in 2‐propanol under the same experimental conditions. These results reveal that the 2‐chloro substituent reduces the hydrogen abstraction ability of the substituted BP triplet, which was not as expected based on the assumption that the electron‐withdrawing group could increase its photoreduction ability. This unusual ortho effect of the chlorine substitution is briefly discussed. Copyright © 2011 John Wiley & Sons, Ltd.