Time-resolved Resonance Raman Study Research Articles

Nonbonded Atomic Contacts Drive Ultrafast Helix Motions in Myoglobin.

The association and dissociation of small ligands regulate the functions of proteins through structural changes in the protein. Such structural changes propagate long distances, and this allostery plays a key role in molecular functions. However, the mechanism by which structural changes are transmitted is poorly understood. Here we show that nonbonded atomic contacts play an essential role in driving the displacement of a helix in picosecond time scale primary structural changes following the dissociation of carbon monoxide from the heme group in myoglobin. The present time-resolved ultraviolet resonance Raman study revealed that the amplitude of this helix displacement was reduced upon substitution of Val68, which contacts the heme in wild-type myoglobin, with a less bulky side chain (Ala). Our findings provided the first direct evidence that structural changes are transmitted not only by covalent bonds, salt bridges and hydrogen bonds but also by nonbonded atomic contacts in the primary protein response upon ligand dissociation. Furthermore, the present results indicate the importance of dense atomic packing in a protein structure for responding to the association and dissociation of small molecules. The high compactness of protein structures makes possible the propagation of structural changes, providing useful clues to the design of molecular machines.

Solvent effects on the structure of the triplet excited state of xanthone: a time-resolved resonance Raman study

Solvent effects, especially intermolecular hydrogen bonding, play a central role in the photophysics and photochemistry of aromatic ketones. To gain insight into the solute–solvent interactions and their implications for structure and reactivity, we studied xanthone (XT) in two different solvents of similar dipolarity: acetonitrile (ACN; aprotic) and methanol (MeOH; protic), using time-resolved resonance Raman (TR3) spectroscopy in conjunction with time-dependent density functional theory calculations. Raman excitation profiles of XT in ACN followed the triplet-triplet absorption band with a shoulder at the blue end, but for MeOH, they followed the triplet-triplet absorption band quite closely; therefore, we propose that the resonance enhancement of Raman peaks are from two states in ACN and from a single state in the MeOH solvent. Furthermore, a resonance Raman peak at 614 cm−1 (a2 symmetry) that appeared in ACN but not in the MeOH solvent has been identified as a vibronic active mode that could be involved in coupling the two lowest 13ππ* (13A1) and 13nπ* (13A2) excited states. This was further confirmed by depolarization ratio measurements of some of the representative TR3 peaks in ACN, which showed a depolarized intensity for the 614 cm−1 peak while the other peaks were polarized. Interestingly, we also observed blue shifting of some of the vibrational frequencies of XT in the 13ππ* state compared with the ground state with increasing solvent polarity. This anomalous blue shift casts doubt on the general use of the resonance canonical structure to explain the structure of the excited states. In summary, we propose that the different hydrogen bonding mechanisms exhibited by the two lowest triplet states of XT separate them further in energy and that this can contribute to its low reactivity towards H atom abstraction in protic solvents. Copyright © 2016 John Wiley & Sons, Ltd.

Direct Observation of Thermal Equilibrium of Excited Triplet States of 9,10-Phenanthrenequinone. A Time-Resolved Resonance Raman Study.

The photochemistry of aromatic ketones plays a key role in various physicochemical and biological processes, and solvent polarity can be used to tune their triplet state properties. Therefore, a comprehensive analysis of the conformational structure and the solvent polarity induced energy level reordering of the two lowest triplet states of 9,10-phenanthrenequinone (PQ) was carried out using nanosecond-time-resolved absorption (ns-TRA), time-resolved resonance Raman (TR(3)) spectroscopy, and time dependent-density functional theory (TD-DFT) studies. The ns-TRA of PQ in acetonitrile displays two bands in the visible range, and these two bands decay with similar lifetime at least at longer time scales (μs). Interestingly, TR(3) spectra of these two bands indicate that the kinetics are different at shorter time scales (ns), while at longer time scales they followed the kinetics of ns-TRA spectra. Therefore, we report a real-time observation of the thermal equilibrium between the two lowest triplet excited states of PQ, assigned to nπ* and ππ* of which the ππ* triplet state is formed first through intersystem crossing. Despite the fact that these two states are energetically close and have a similar conformational structure supported by TD-DFT studies, the slow internal conversion (∼2 ns) between the T(2)(1(3)nπ*) and T(1)(1(3)ππ*) triplet states indicates a barrier. Insights from the singlet excited states of PQ in protic solvents [ J. Chem. Phys. 2015 , 142 , 24305 ] suggest that the lowest nπ* and ππ* triplet states should undergo hydrogen bond weakening and strengthening, respectively, relative to the ground state, and these mechanisms are substantiated by TD-DFT calculations. We also hypothesize that the different hydrogen bonding mechanisms exhibited by the two lowest singlet and triplet excited states of PQ could influence its ISC mechanism.

Insights into Protein Structure and Dynamics by Ultraviolet and Visible Resonance Raman Spectroscopy.

Raman spectroscopy is a form of vibrational spectroscopy based on inelastic scattering of light. In resonance Raman spectroscopy, the wavelength of the incident light falls within an absorption band of a chromophore, and this overlap of excitation and absorption energy greatly enhances the Raman scattering efficiency of the absorbing species. The ability to probe vibrational spectra of select chromophores within a complex mixture of molecules makes resonance Raman spectroscopy an excellent tool for studies of biomolecules. In this Current Topic, we discuss the type of molecular insights obtained from steady-state and time-resolved resonance Raman studies of a prototypical photoactive protein, rhodopsin. We also review recent efforts in ultraviolet resonance Raman investigations of soluble and membrane-associated biomolecules, including integral membrane proteins and antimicrobial peptides. These examples illustrate that resonance Raman is a sensitive, selective, and practical method for studying the structures of biological molecules, and the molecular bonding, geometry, and environments of protein cofactors, the backbone, and side chains.

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.

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.

Time-Resolved Resonance Raman Spectroscopic Studies on the Triplet Excited State of Thioxanthone

Thioxanthone has been investigated extensively owing to its unique photochemical and photophysical applications and its solvatochromic behavior. Here, we report the time-resolved resonance Raman studies on the structure of the lowest triplet excited state of thioxanthone in carbon tetrachloride. In addition, FT-IR and FT-Raman techniques have been used to study the vibrational structure in the ground state. To corroborate the experimental findings, density functional theory calculations have been carried out. Isotopic calculations and normal coordinate analysis have been used to help in assigning the observed bands to Raman vibrational modes. Structural information derived from this study is expected to help in better understanding the triplet state photochemistry of thioxanthone.

Time-Resolved Resonance Raman Studies on Proton-Induced Electron-Transfer Reaction from Triplet Excited State of 2-Methoxynaphthalene to Decafluorobenzophenone

In this paper, time-resolved resonance Raman (TR3) spectra of intermediates generated by proton-induced electron-transfer reaction between triplet 2-methoxynaphthalene ((3)ROMe) and decafluorobenzophenone (DFBP) are presented. The TR3 vibrational spectra and structure of 2-methoxynaphthalene cation radical (ROMe(•+)) have been analyzed by density functional theory (DFT) calculation. It is observed that the structure of naphthalene ring of ROMe(•+) deviates from the structure of cation radical of naphthalene.

Time-Resolved Resonance Raman Study of the Effect of pH on the Photoreactions of 3-Benzoylpyridine in Aqueous Solution

A nanosecond time-resolved resonance Raman investigation of the photoreactions of 3-benzoylpyridine (3-BPy) in different pH aqueous solutions is reported. In neutral, basic, and pH = 5 aqueous solution conditions, the photoreduction reaction from the triplet 3-BPy species is observed to produce the corresponding 3-phenyl pyridyl ketyl radical that was also observed in a 2-propanol solvent. Under moderate acidic conditions (at pH = 3 for example), most of the 3-BPy triplet state species goes through two protonation steps at the nitrogen atom and the carbonyl oxygen atom after UV laser photolysis and then forms a short-lived hydration intermediate via a hydration reaction at the ortho position in the benzene ring. This new species is tentatively assigned to the o-3[3-BPyH+.H2O] hydration species. In acidic aqueous solutions with a pH < or = 1, the protonated triplet states of 3-BPy cations at the nitrogen atom are generated from photoexcitation of the protonated ground state and are subsequently further protonated at the carbonyl oxygen atom to form a 3-BPy-dication triplet state. This dication intermediate reacts with water molecules at the ortho position of the benzene ring to produce the o-3[3-BPyH+.H2O] hydration species. The mechanisms of photoreduction observed for 3-BPy in different pH aqueous solutions were investigated using density functional theory calculations, and these results were used to help assign the intermediates observed in the experiments. The structures and properties of these species are briefly discussed, and an overall photoreaction mechanism is proposed based on the results from the time-resolved resonance Raman experiments and the density functional theory calculations.

Influence of Solvent on Photoinduced Electron-Transfer Reaction: Time-Resolved Resonance Raman Study

Time-resolved resonance Raman spectroscopy (TR3) has been used to study the effect of solvent polarity on the mechanism and nature of intermediates formed in photoinduced electron-transfer reaction between triplet flouranil ((3)FL) and tetramethylbenzene (TMB). Comparison of the TR3 spectra in polar, nonpolar, and medium polar media suggests that formation of radical anion due to electron-transfer reaction between (3)FL and TMB is favored in more polar solvents, whereas ketyl radical formation is more favored in less polar media. Compared to ketyl radical, the extent of radical anion formation is negligible in nonpolar solvents. Therefore, it is inferred that in nonpolar media ketyl radical is mainly generated by hydrogen-transfer reaction in the encounter complex between (3)FL and TMB. In solvents of medium polarity, the ion-pair decay leads to the formation of both ketyl radical and ketyl radical formed from the encounter between triplet state and the donor. Thus, competition between the formation of ketyl radical and ion pair is influenced by the solvent polarity. The nature of the ion pair in different solvent polarity has been investigated from the changes observed in the vibrational frequency of (fluoranil) FL part of the complex.

Time-Resolved Resonance Raman and Density Functional Theory Investigation of the Photoreactions of Benzophenone in Aqueous Solution

A time-resolved resonance Raman study of the photoreactions of benzophenone (BP) in neutral, alkaline, and acidic aqueous solutions is reported. Under neutral and alkaline conditions, a hydrogen abstraction reaction is observed to form the corresponding diphenyl ketyl (DPK) radical with different reaction rates while under acidic condition the protonation of triplet benzophenone occurs simultaneously, which is followed by a faster hydration reaction to produce the corresponding photohydration short-lived intermediates that were observed during experiments and that were tentatively assigned to the m-(3)BP x H(2)O and the o-(1)BP x H(2)O species. The dynamics, structures, and properties of these species are briefly discussed.

Reaction of the 4-Biphenylnitrenium Ion with 4-Biphenyl Azide to Produce a 4,4′-Azobisbiphenyl Stable Product: A Time-Resolved Resonance Raman and Density Functional Theory Study

A time-resolved resonance Raman (TR(3)) and density functional theory (DFT) study of the reaction of the 4-biphenylnitrenium ion with 4-biphenyl azide in a mixed aqueous solution is reported. The reaction of the 4-biphenylnitrenium ion with its unphotolyzed precursor 4-biphenyl azide in a mixed aqueous solution generates a 4,4'-azobisbiphenyl stable product via an intermediate species. With the aid of DFT calculations for likely transient species, this intermediate was tentatively assigned to a 4,4'-azobisbiphenyl cation. The DFT calculations predict this reaction can take place via two pathways that compete with one another to produce the trans and cis 4,4'-azobisbiphenyl product. The observation of the 4,4'-azobisbiphenyl cation intermediate demonstrates that the reaction of the arylnitrenium ion with its aryl azide to produce a stable azo product occurs via a stepwise mechanism.

Time-Resolved Resonance Raman Observation of the Dimerization of Didehydroazepines in Solution

Time-resolved resonance Raman (TR3) studies of the photochemistry of phenyl azide, 3-hyroxyphenyl azide, 3-methoxyphenyl azide and 3-nitrophenyl azide in acetonitrile:water solutions is reported. After photolysis of these four aryl azides in room temperature solutions, only one species was observed in the TR3 spectra for each azide, respectively at the probe wavelengths employed in the TR3 experiments. The species observed after photolysis of 3-nitrophenyl azide was assigned to 3,3'-dinitroazobenzene, an azo compound formed from the dimerization reaction of triplet 3-nitrophenylnitrene. In contrast, the species observed after photolysis of phenyl azide, 3-hydroxyphenyl azide and 3-methoxyphenyl azide were tentatively assigned to intermediates formed from the dimerization of didehydroazepines that are produced from the ring expansion reaction of the respective singlet arylnitrene. To our knowledge, this is the first time-resolved vibrational spectroscopic observation of the dimerization reaction of didehydroazepines in solution. In addition, these are the first resonance Raman spectra reported for dimers formed from didehydroazepines. We briefly discuss the structures, properties and chemical reactivity of the dimer species observed in the TR3 spectra and possible implications for the photochemistry of aryl azides.

Femtosecond Transient Absorption and Nanosecond Time‐Resolved Resonance Raman Study of the Solvent‐Dependent Photo‐Deprotection Reaction of Benzoin Diethyl Phosphate

A combined femtosecond transient absorption (fs-TA) and nanosecond time-resolved resonance Raman (ns-TR3) study was performed to directly detect the dynamics and elucidate the mechanism of the excited state deactivation and solvent-dependent photo-deprotection pathways for benzoin diethyl phosphate (BDP) in neat acetonitrile (MeCN) and 75 % H2O/25 % MeCN. Comparison of the TA spectral evolution observed in the two solvents provides explicit evidence that the photophysical deactivation of the BDP singlet excited state has little solvent dependence. The TA spectra also indicate the related internal conversion (IC) and intersystem crossing (ISC) processes occur rapidly on hundreds of femtoseconds and approximately 2-3 ps time scales, respectively. From this and in conjunction with a photochemistry study and ground state resonance Raman (RR) measurements, the TA results reveal that the phenacyl localized BDP triplet state (that is mainly npi* nature) is the common and immediate precursor to the photo-deprotection reaction in both solvents. However, the triplet deprotection follows different pathways in neat MeCN versus the largely water containing solvent. The deprotection reaction in MeCN was determined to occur with a approximately 11 ns time constant and the reaction was found to be an unimolecular process leading to elimination of the diethyl phosphoric acid apparently concurrent with cyclization to yield the benzofuran product. In the water mixed solvent, the triplet reaction was observed to proceed with a approximately 15 ns time constant and the reaction leads to not only the deprotection-cyclization but also a heterolytic dissociation to release the diethyl phosphate anion through a branching and competing mechanism. The ns-TR3 spectra combined with relevant DFT calculations have been used to characterize the dynamics, structure and vibrational frequencies to help identify the important intermediates as well as to explore the reaction pathway leading to formation of the solvolysis product in the largely water solvent. A consecutive mechanism has been revealed for the heterolysis-solvolysis reaction in the water mixed solvent. The present work provides direct and irrevocable evidence for the dynamics and mechanistic description of the overall photophysics and deprotection related photochemistry for BDP.

Time-Resolved Resonance Raman and Density Functional Theory Study of the Deprotonation Reaction of the Triplet State of p-Hydroxyacetophenone in Water Solution

[reaction; see text] Picosecond and nanosecond time-resolved resonance Raman (TR(3)) spectroscopy was employed to investigate the deprotonation/ionization reaction of p-hydroxyacetophenone (HA) after ultraviolet photolysis in water solution. The TR(3) spectra in conjunction with density functional theory (DFT) calculations were used to characterize the structure and dynamics of the excited-state HA deprotonation to form HA anions in near neutral water solvent. DFT calculations based on a solute-solvent intermolecular H-bonded complex model containing up to three water molecules were used to evaluate the H-bond interactions and their influence on the deprotonation reaction and the structures of the intermediates. The deprotonation reaction was found to occur on the triplet manifold with a planar H-bonded HA triplet complex as the precursor species. The HA triplet species is generated within several picoseconds and then decays with a approximately 10 ns time constant to produce the HA triplet anion species after 267 nm photolysis of HA in water solution. The triplet anion species was observed to decay with a time constant of about 90 ns into the ground-state anion species that was found to have a lifetime of about 200 ns. The DFT calculations on the H-bonded complexes of the anion triplet and ground-states species suggest that these anion species are H-bonded complexes with planar quinonoidal structures containing two water molecules H-bonded, respectively, with oxygen lone pairs of the carbonyl and deprotonated hydroxyl moieties. A deactivation scheme of the photoexcited HA in regard to the deprotonation reaction in neutral water solutions was proposed. With the above dynamic and structural information available, we briefly discuss the possible implications of the model HA photochemistry in water solutions for the photodeprotection reactions of related p-HP phototrigger compounds in aqueous solutions.

Time-Resolved Resonance Raman and Density Functional Theory Study of Hydrogen-Bonding Effects on the Triplet State of p-Methoxyacetophenone

Picosecond and nanosecond time-resolved resonance Raman spectroscopy combined with density functional theory calculations have been performed to characterize the structure, dynamics, and hydrogen-bonding effects on the triplet state of the phototrigger model compound p-methoxyacetophenone (MAP) in cyclohexane, MeCN, and 50% H2O/50% MeCN (v:v) mixed solvent. Analogous work has also been done to study the corresponding ground state properties. The ground and triplet states of MAP were both found to be associated strongly with the water solvent molecules in the 50% H2O/50% MeCN solvent system. A hydrogen-bond complex model involving one or two water molecules bonded with the oxygen atoms of the MAP carbonyl and methoxy moieties has been employed to explore the hydrogen-bond interactions and their influence on the geometric and electronic properties for the ground and triplet states of MAP. Among the various hydrogen-bond configurations examined, the carbonyl hydrogen-bond configuration involving one water molecule was calculated to lead to the most stable hydrogen-bond complex for both the ground and the triplet states with the strength of the hydrogen-bond interaction being stronger in the triplet state than the ground state. The increased carbonyl located hydrogen-bond strength in the triplet state results in substantial modification of both the electronic and the structural conformation so that the triplet of the hydrogen-bond complex can be considered as a distinct species from the free MAP triplet state. This provides a framework to interpret the differences observed in the TR3 spectral and triplet lifetime obtained in the neat MeCN solvent (attributed to the free MAP triplet state) and the 50% H2O/50% MeCN solvent (due to the triplet of the hydrogen-bond complex). Temporal evolution at early picosecond times indicates rapid ISC conversion, and subsequent relaxation of the excess energy of the initially formed energetic triplets occurs for both the free MAP and the hydrogen-bond complex. The triplet of the carbonyl hydrogen-bond complex appears to be generated directly from the corresponding ground state complex and it does not dissociate back to the free triplet state within the triplet state lifetime. We briefly discuss the influence of the carbonyl hydrogen-bond effect on the pi pi* triplet reactivity for MAP and closely related compounds.

Direct Observation of the 4-Methoxyphenylnitrene Intersystem Crossing from S1 to T1 Using Picosecond Kerr-Gated Time-Resolved Resonance Raman Spectroscopy

A picosecond Kerr-gated time-resolved resonance Raman (ps-KTR(3)) study of the singlet 4-methoxyphenylnitrene intersystem crossing to produce the triplet 4-methoxyphenylnitrene species is reported. The experimental resonance Raman vibrational band frequencies observed for the singlet and triplet 4-methoxylphenylnitrene species in the time-resolved Raman experiments are compared to each other and to predictions from previously published density functional theory calculations. The structure, properties, and chemical reactivity of the singlet and triplet states of the 4-methoxyphenylnitrenes are briefly discussed.

2P100 グルコース酸化酵素の光誘起電子移動反応 : ピコ秒時間分解紫外共鳴ラマン分光法による研究(蛋白質 E) 計測・解析の方法論))

2P100 グルコース酸化酵素の光誘起電子移動反応 : ピコ秒時間分解紫外共鳴ラマン分光法による研究(蛋白質 E) 計測・解析の方法論))

3P064 温度ジャンプ・時間分解共鳴ラマン分光によるシトクロムc熱変性初期過程の研究(蛋白質 C) 物性(安定性、折れたたみなど)))

3P064 温度ジャンプ・時間分解共鳴ラマン分光によるシトクロムc熱変性初期過程の研究(蛋白質 C) 物性(安定性、折れたたみなど)))

Vibrational excitation after ultrafast intramolecular proton transfer of TINUVIN: a time-resolved resonance Raman study

Vibrational excitation and energy redistribution after the ultrafast intramolecular proton transfer cycle of TINUVIN was investigated by picosecond pump–probe resonance Raman spectroscopy. Our studies demonstrate that the line shape function of the resonance Raman cross section dramatically changes due to vibrational excitation. Therefore, instead of determining vibrational populations directly from anti-Stokes resonance Raman kinetics, excitation of the modes has been characterized by their time-dependent ratios of anti-Stokes and Stokes resonance Raman intensities. Using this novel approach it is shown that a low-frequency proton transfer promoting mode serves as a major accepting mode of the proton transfer cycle.