Hole-burning Spectroscopy Research Articles

Conformational structures of 3-cyanoindole-(H2O)n (n = 0–2) by UV–UV hole-burning and IR-dip spectroscopy

The excitation spectra of 3-cyanoindole (3CI) and its water clusters, 3CI-(H2O)n (n=1 and 2), have been measured by mass-selected resonant two-photon ionization and UV–UV hole-burning (UVHB) spectroscopy. The observed spectra are compared with the predictions of ab initio and density functional theory calculations. The comparison suggests that the spectra arise from a single conformer of 3CI and two conformers of 3CI-(H2O)n (n=1 and 2) each. UVHB and IR-dip spectroscopy were employed to identify the conformers of 3CI-(H2O)1 and 3CI-(H2O)2. The conformation-specific structural assignment of the conformers is compared and discussed with theoretical calculations.

New Method for Double-Resonance Spectroscopy in a Cold Quadrupole Ion Trap and Its Application to UV–UV Hole-Burning Spectroscopy of Protonated Adenine Dimer

A novel method for double-resonance spectroscopy in a cold quadrupole ion trap is presented, which utilizes dipolar resonant excitation of fragment ions in the quadrupole ion trap. Photofragments by a burn laser are removed by applying an auxiliary RF to the trap, and a probe laser detects the depletion of photofragments by the burn laser. By scanning the wavelength of the burn laser, conformation-specific UV spectrum of a cold ion is obtained. This simple and powerful method is applicable to any type of double-resonance spectroscopy in a cold quadrupole ion trap and was applied to UV-UV hole-burning spectroscopy of protonated adenine dimer. It was found that protonated adenine dimer has multiple conformers/tautomers, each with multiple excited states with drastically different excited state dynamics.

Charge-transfer character of the low-energy Chl a Q(y) absorption band in aggregated light harvesting complexes II.

One of the key functions of the major light harvesting complex II (LHCII) of higher plants is to protect Photosystem II from photodamage at excessive light conditions in a process called "non-photochemical quenching" (NPQ). Using hole-burning (HB) spectroscopy, we investigated the nature of the low-energy absorption band in aggregated LHCII complexes - which are highly quenched and have been established as a good in vitro model for NPQ. Nonresonant holes reveal that the lowest energy state (located near 683.3 nm) is red-shifted by ~4 nm and significantly broader (by a factor of 4) as compared to nonaggregated trimeric LHCII. Resonant holes burned in the low-energy wing of the absorption spectrum (685-710 nm) showed a high electron-phonon (el-ph) coupling strength with a Huang-Rhys factor S of 3-4. This finding combined with the very low HB efficiency in the long-wavelength absorption tail is consistent with a dominant charge-transfer (CT) character of the lowest energy transition(s) in aggregated LHCII. The value of S decreases at shorter wavelengths (<685 nm), in agreement with previous studies (J. Pieper et al., J. Phys. Chem. B 1999, 103, 2422-2428), proving that the low-energy excitonic state is strongly mixed with the CT states. Our findings support the mechanistic model in which Chl-Chl CT states formed in aggregated LHCII are intermediates in the efficient excited state quenching process (M. G. Müller et al., Chem. Phys. Chem. 2010, 11, 1289-1296; Y. Miloslavina et al., FEBS Lett. 2008, 582, 3625-3631).

On the Controversial Nature of the 825 nm Exciton Band in the FMO Protein Complex.

The nature of the low-energy 825 nm band of the Fenna-Matthews-Olson (FMO) protein complex from Chlorobaculum tepidum at 5 K is discussed. It is shown, using hole-burning (HB) spectroscopy and excitonic calculations, that the 825 nm absorption band of the FMO trimer cannot be explained by a single electronic transition or overlap of electronic transitions of noninteracting pigments. To explain the shape of emission and nonresonant HB spectra, downward uncorrelated excitation energy transfer (EET) between trimer subunits should be taken into account. Modeling studies reveal the presence of three sub-bands within the 825 nm band, in agreement with nonresonant HB and emission spectra. We argue that after light induced coherences vanish, uncorrelated EET between the lowest exciton levels of each monomer takes place. HB induced spectral shifts provide a new insight on the energy landscape of the FMO protein.

Mimicking the First Turn of an α-Helix with an Unnatural Backbone: Conformation-Specific IR and UV Spectroscopy of Cyclically Constrained β/γ-Peptides

The folding preferences of two capped, constrained β/γ-dipeptide isomers, Ac-βACPC-γACHC-NHBn and Ac-γACHC-βACPC-NHBn, (designated βγ and γβ, respectively), have been investigated using single- and double-resonance ultraviolet and infrared spectroscopy in the gas phase. These capped β/γ-dipeptides have the same number of backbone atoms between their N- and C-termini as a capped α-tripeptide and thus serve as a minimal structural unit on which to test their ability to mimic the formation of the first turn of an α-helix. Resonant two-photon ionization and UV-UV hole-burning spectroscopy were performed in the S0-S1 region, revealing the presence of three unique conformations of βγ and a single conformation of γβ. Resonant ion-dip infrared spectra were obtained in the NH stretch region from 3300 to 3500 cm(-1) and in both the amide I and amide II regions from 1400 to 1800 cm(-1). These infrared spectra were compared to computational predictions from density functional theory calculations at the M05-2X/6-31+G(d) level, leading to assignments for the observed conformations. Two unique bifurcated C8/C13 H-bonded ring structures for βγ and a single bifurcated C9/C13 H-bonded ring structure for γβ were observed. In all cases, the H-bonding patterns faithfully mimic the first full turn of an α-helix, most notably by containing a 13-membered H-bonded cycle but also by orienting the interior amide group so that it is poised to engage in a second C13 H-bond as the β/γ-peptide lengthens in size. The structural characteristics of the β/γ-peptide version of the 13-helix turn are compared with the α-helix counterpart and with a reported crystal structure for a longer β/γ-peptide oligomer.

Fast Nonradiative Decay in o-Aminophenol

The gas phase structure of 2-aminophenol has been investigated using UV-UV as well as IR-UV hole burning spectroscopy. The presence of a free OH vibration in the IR spectrum rules out the contribution of the cis isomer, which is expected to have an intramolecular H-bond, to the spectra. The excited state lifetimes of different vibronic levels have been measured with pump-probe picosecond experiments and are all very short (35 ± 5) ps as compared to other substituted phenols. The electronic states and active vibrational modes of the cis and trans isomers have been calculated with ab initio methods for comparison with the experimental spectra. The Franck-Condon simulation of the spectrum using the calculated ground and excited state frequencies of the trans isomer is in good agreement with the experimental one. The very short excited state lifetime of 2-aminophenol can then be explained by the strong coupling between the two first singlet excited states due to the absence of symmetry, the geometry of the trans isomer being strongly nonplanar in the excited state.

UV-excitation from an experimental perspective: frequency resolved.

Electronic spectroscopy of DNA bases in the gas phase provides detailed information about the electronic excitation, which places the molecule in the Franck-Condon region in the excited state and thus prepares the starting conditions for excited-state dynamics. Double resonance or hole-burning spectroscopy in the gas phase can provide such information with isomer specificity, probing the starting potential energy landscape as a function of tautomeric form, isomeric structure, or hydrogen bonded or stacked cluster structure. Action spectroscopy, such REMPI, can be affected by excited-state lifetimes.

Single-conformation UV and IR spectroscopy of model G-type lignin dilignols: the β–O–4 and β–β linkages

Single-conformation ultraviolet and infrared spectroscopy was performed on dilignols containing two of the three biologically prevalent β-lignol linkages, erythro β–O–4 (β-aryl ether) and (±) β–β (pinoresinol). Both dilignols contain guaiacol(G)-type sub-units, representative of these linkages in G-type lignin. Resonant two-photon ionization (R2PI), IR-UV, and UV-UV holeburning (UVHB) spectroscopy in the cold, isolated environment of a supersonic expansion was carried out to determine the spectroscopic signatures associated with each linkage conformation, revealing striking differences in the vibronic intensity patterns between the two molecules in the UV. Two conformational isomers were found for the β–O–4 dilignol, both being classified into the fully hydrogen-bonded family with α-OH⋯OCH3 (C8) and γ-OH⋯Oβ (C5) H-bonds that are characteristic of the β–O–4 linkage. Conversely, a single dominant conformation was found for the conformationally-constrained pinoresinol. Resonant ion-dip infrared (RIDIR) spectroscopy provided conformation-specific IR spectra in the OH stretch and alkyl CH stretch regions, yielding complementary data that reported on both the intramolecular H-bonding and more subtle linkage features, respectively. DFT M05-2X calculations predict that the rigid β–β linkage had far fewer low-energy conformations in the first 20 kJ mol−1 (3) than the more flexible β–O–4 linkage (45). In the β–O–4 lignin dimer, the distinct UV chromophores lead to a splitting between S1 and S2 states that is determined mainly by the differences in the chemical structures of the two chromophores. In pinoresinol however, the assigned structure has C2 symmetry, with a calculated vertical excitonic splitting between the S1 and S2 states of 74 cm−1 (TDDFT). After taking into account the reduction in splitting associated with the geometry change in the aromatic rings upon electronic excitation, a vibronically quenched excitonic splitting of no more than a few wavenumbers is predicted for the C2 symmetric pinoresinol, but a definite experimental confirmation was not possible. These results predict that, under most circumstances, adjacent chromophores along a lignin polymer chain are not significantly electronically coupled to one another, and can be treated largely as isolated chromophores.

Low-temperature spectroscopy of bacteriochlorophyll c aggregates

Chlorosomes from green photosynthetic bacteria belong to the most effective light-harvesting antennas found in nature. Quinones incorporated in bacterichlorophyll (BChl) c aggregates inside chlorosomes play an important redox-dependent photo-protection role against oxidative damage of bacterial reaction centers. Artificial BChl c aggregates with and without quinones were prepared. We applied hole-burning spectroscopy and steady-state absorption and emission techniques at 1.9 K and two different redox potentials to investigate the role of quinones and redox potential on BChl c aggregates at low temperatures. We show that quinones quench the excitation energy in a similar manner as at room temperature, yet the quenching process is not as efficient as for chlorosomes. Interestingly, our data suggest that excitation quenching partially proceeds from higher excitonic states competing with ultrafast exciton relaxation. Moreover, we obtained structure-related parameters such as reorganization energies and inhomogeneous broadening of the lowest excited state, providing experimental ground for theoretical studies aiming at designing plausible large-scale model for BChl c aggregates including disorder.

Structure and Hydrogen-Bonding Ability of Estrogens Studied in the Gas Phase

The structures of estrogens (estrone(E1), β-estradiol(E2), and estriol(E3)) and their 1:1 hydrogen-bonded (hydrated) clusters with water formed in supersonic jets have been investigated by various laser spectroscopic methods and quantum chemical calculations. In the S1-S0 electronic spectra, all three species exhibit the band origin in the 35,050-35,200 cm(-1) region. By use of ultraviolet-ultraviolet hole-burning (UV-UV HB) spectroscopy, two conformers, four conformers, and eight conformers, arising from different orientation of OH group(s) in the A-ring and D-ring, are identified for estrone, β-estradiol, and estriol, respectively. The infrared-ultraviolet double-resonance (IR-UV DR) spectra in the OH stretching vibration are observed to discriminate different conformers of the D-ring OH for β-estradiol and estriol, and it is suggested that in estriol only the intramolecular hydrogen bonded conformer exists in the jet. For the 1:1 hydrated cluster of estrogens, the S1-S0 electronic transition energies are quite different depending on whether the water molecule is bound to A-ring OH or D-ring OH. It is found that the water molecule prefers to form an H-bond to the A-ring OH for estrone and β-estradiol due to the higher acidity of phenolic OH than that of the alcoholic OH. On the other hand, in estriol the water molecule prefers to be bound to the D-ring OH due to the formation of a stable ring-structure H-bonding network with two OH groups. Thus, the substitution of one hydroxyl group to the D-ring drastically changes the hydrogen-bonding preference of estrogens.

Fluorescence excitation spectrum and solvent-assisted conformational isomerization (SACI) of jet-cooled acetaminophen

Fluorescence excitation spectrum of jet-cooled acetaminophen was obtained. When AAP was expanded with a buffer gas containing 0.3–1.1% of water, absorption peaks of the less stable trans conformer was significantly reduced by solvent-assisted conformational isomerization (SACI), which is confirmed by a separately measured UV–UV hole burning spectroscopy. It is also confirmed by quantum mechanical calculation and RRKM calculation that it is energetically and kinetically possible to induce SACI in AAP with water. The SACI mechanism suggests a possible pathway that acetaminophen can adopt an active conformation in vivo, which is need for molecular recognition and drug activity.

Conformational Heterogeneity of Methyl 4-Hydroxycinnamate: A Gas-Phase UV–IR Spectroscopic Study

UV excitation and IR absorption spectroscopy on jet-cooled molecules is used to study the conformational heterogeneity of methyl 4-hydroxycinnamate, a model chromophore of the Photoactive Yellow Protein (PYP), and to determine the spectroscopic properties of the various conformers. UV-UV depletion spectroscopy identifies four different species with distinct electronic excitation spectra. Quantum chemical calculations argue that these species are associated with different conformers involving the s-cis/s-trans configuration of the ester with respect to the propenyl C-C single bond and the syn/anti orientation of the phenolic OH group. IR-UV hole-burning spectroscopy is used to record their IR absorption spectra in the fingerprint region. Comparison with IR absorption spectra predicted by quantum chemical calculations provides vibrational markers for each of the conformers, on the basis of which each of the species observed with UV-UV depletion spectroscopy is assigned. Although both DFT and wave function methods reproduce experimental frequencies, we find that calculations at the MP2 level are necessary to obtain agreement with experimentally observed intensities. To elucidate the role of the environment, we compare the IR spectra of the isolated conformers with IR spectra of methyl 4-hydroxycinnamate-water clusters, and with IR spectra of methyl 4-hydroxycinnamate in solution.

Towards a first-principles model of Fermi resonance in the alkyl CH stretch region: Application to 1,2-diphenylethane and 2,2,2-paracyclophane

The spectroscopy of two flexible hydrocarbons, 1,2-diphenylethane (DPE) and 2,2,2-paracyclophane (TCP) is presented, and a predictive theoretical model for describing the alkyl CH stretch region of these hydrocarbons is developed. Ultraviolet hole-burning spectroscopy identified two isomers of DPE and a single conformation of TCP present in the supersonic jet expansion. Through the analysis of the ground state low-frequency vibronic spectroscopy obtained by dispersed fluorescence, conformational assignments were made for both DPE and TCP. The two isomers of DPE were found to retain the low energy structures of butane, being present in both the gauche and anti structures. TCP forms a C(2) symmetric structure, differing from the predicted lower energy C(3) conformation by the symmetry of the ethano bridges (-CH(2)CH(2)-) linking the phenyl substituents. Resonant ion-dip infrared spectroscopy is used to record single-conformation IR spectra of the two conformers of DPE and the single conformer of TCP in the alkyl CH stretch region and in the mid-IR that covers the CH bend fundamentals. A local mode Hamiltonian that incorporates cubic stretch-bend coupling is developed. Its parameters are obtained from density functional theory methods. Full dimensional calculations are compared to those that use reduced dimensional Hamiltonians in which anharmonic CH stretches and scissor modes are Fermi coupled. Excellent agreement is found. Scale factors of select terms in the reduced dimensional Hamiltonian are determined by fitting the theoretical Hamiltonian to the anti-DPE spectrum. The scaled Hamiltonian is then used to predict successfully structures for the remaining lower symmetry experimentally determined spectra in the alkyl CH stretch region.

Conformationally resolved spectra of acetaminophen by UV-UV hole burning and IR dip spectroscopy in the gas phase

Electronic and vibrational spectra of acetaminophen were measured by using UV-UV hole burning (HB) and IR dip spectroscopy. HB spectra show the coexistence of 4 different species, which include two new ones. Low-frequency transitions in the spectra are reproduced by a one-dimensional periodic potential with a free-rotor basis set for the methyl group. From the analysis, we concluded that acetaminophen has two conformers and each conformer gives two independent transitions starting from the most stable 0a(1) and the hot 1e internal rotational levels. It is also found that the HB spectrum of the trans-conformer in the previous report is that from the 1e excited level, while the HB spectrum of the cis-conformer is contaminated by the transitions of the trans-conformer. Potential curves of the methyl rotational motion are determined both in S(0) and S(1).

Laser Spectroscopic Study of Cold Host–Guest Complexes of Crown Ethers in the Gas Phase

A laser spectroscopic study on the structure and dynamics of cold host-guest inclusion complexes of crown ethers (CEs) with various neutral and ionic species in the gas phase is presented. The complexes with neutral guest species are formed by using supersonic free jets, and those with ionic species are generated with electrospray ionization combined with a cold 22-pole ion trap. For CEs, various sizes of 3n-crown-n ethers (n=4, 5, 6, and 8) and their benzene-substituted species are used. For the guest species, water, methanol, ammonia, acetylene, and phenol are employed as neutral guest species, and for charged guest species, alkali metal cations are chosen. The electronic and vibrational spectra of the complexes are measured by using various laser spectroscopic methods; electronic spectra for the neutral complexes are measured by laser-induced fluorescence. Discrimination of different species such as conformers is performed by ultraviolet-ultraviolet hole-burning spectroscopy. The vibrational spectra of selected species are observed by infrared-ultraviolet double-resonance (IR-UV DR) spectroscopy. For the ionic complexes, ultraviolet photodissociation and IR-UV DR spectroscopy are applied. The complex structures are determined by comparing the observed spectra with those of possible structures obtained by density functional theory calculations. How the host CEs change their conformation or which conformer prefers to form unique inclusion complexes are discussed. These results reveal the key interactions for forming special complexes leading to molecular recognition.

Structure of Indole···Imidazole Heterodimer in a Supersonic Jet: A Gas Phase Study on the Interaction between the Aromatic Side Chains of Tryptophan and Histidine Residues in Proteins

In this study, we have investigated the binding motifs between the aromatic side chains of tryptophan and histidine residues in proteins by studying the indole···imidazole heterodimer in a supersonic jet. Different spectroscopic techniques including resonant two-photon ionization (R2PI), UV-UV hole-burning, and resonant ion dip infrared (RIDIR) spectroscopy merged with quantum chemistry calculations have been used for this work. UV-UV hole-burning spectroscopy has been used to confirm the presence of only one structure of the dimer in the experiment. From the comparison of the RIDIR spectrum of the observed dimer with the theoretical IR spectra of different structures of the dimer, it is found that the dimer present in the experiment has a V-shaped structure held by N-H···N hydrogen bond, C-H···π, and weakly present π···π stacking interactions. The most important finding of the present study is that the noncovalent interactions present in the observed dimer have a close resemblance with those present between tryptophan and histidine residues in a nonfluorescent flavoprotein. The present spectroscopic investigation on the indole···imidazole dimer has also immense pharmaceutical significance as this imparts molecular level understanding about the binding motifs of the imidazole drugs with the indole chromophore present in proteins.

Ultraviolet–Ultraviolet Hole Burning Spectroscopy in a Quadrupole Ion Trap: Dibenzo[18]crown‐6 Complexes with Alkali Metal Cations

Conformation selective: A new technique of ultraviolet-ultraviolet hole burning spectroscopy that can be applied to ions stored in a quadrupole ion trap (QIT) is developed and used to obtain the conformation-selective electronic spectra of dibenzo[18]crown-6 complexes with alkali metal cations (M(+), see picture; F(+) = fragment).

Probing Electron-Transfer Times in Photosynthetic Reaction Centers by Hole-Burning Spectroscopy.

A brief discussion is presented of transient hole-burned (HB) spectra (and the information that they provide) obtained for isolated reaction centers (RCs) from wild-type (WT) Rhodobacter sphaeroides, RCs containing zinc-bacteriochlorophylls (Zn-BChls), and RCs of Photosystem II (PSII) from spinach and Chlamydomonas reinhardtii . The shape of the spectral density and the strength of electron-phonon coupling in bacterial RCs are discussed. We focus, however, on heterogeneity of isolated PS II RCs from spinach and, in particular, Chlamydomonas reinhardtii , site energies of active (electron acceptor) and inactive pheophytins, the nature of the primary electron donor(s), and the possibility of multiple charge-separation (CS) pathways in the isolated PSII RC. We conclude with comments on current efforts in HB spectroscopy in the area of photosynthesis and future directions in HB spectroscopy.

Hole-Burning Spectroscopy as a Probe of Nano-environments and Processes in Biomolecules: A Review

Hole-burning spectroscopy, a high-resolution spectroscopic technique, allows details of heterogeneous nano-environments in biological systems to be obtained from broad absorption bands. Recently, this technique has been applied to proteins, nucleic acids, cells, and substructures of water to probe the electrostatic conditions created by macromolecules and the surrounding solvent. Starting with the factors that obscure the homogeneous linewidth of a chromophore within an inhomogeneously broadened absorption or emission band, we describe properties and processes in biological systems that are reflected in the measured hole spectra. The technique also lends itself to the resolution of perturbation experiments, such as temperature cycling to elucidate energy landscape barriers, applied external electric fields (Stark effect) to measure net internal electric fields, and applied hydrostatic pressure to find the volume compressibility of proteins.

Photochemistry of Benzylallene: Ring-Closing Reactions to Form Naphthalene

Conformer-specific, vibrationally resolved electronic spectroscopy of benzylallene (4-phenyl-1,2-butadiene) is presented along with a detailed analysis of the products formed via its ultraviolet photoexcitation. Benzylallene is the minor product of the recombination of benzyl and propargyl radicals. The mass-selective resonant two-photon ionization spectrum of benzylallene was recorded under jet-cooled conditions, with its S(0)-S(1) origin at 37,483 cm(-1). UV-UV holeburning spectroscopy was used to show that only one conformer was present in the expansion. Rotational band contour analysis provided rotational constants and transition dipole moment direction consistent with a conformation in which the allene side chain is in the anti position, pointing away from the phenyl ring. The photochemistry of benzylallene was studied in a pump-probe geometry in which photoexcitation occurred by counter-propagating the expansion with a photoexcitation laser. The laser was timed to interact with the gas pulse in a short tube that extended the collisional region of the expansion. The products were cooled during expansion of the gas mixture into vacuum, before being interrogated using mass-selective resonant two-photon ionization. The UV-vis spectra of the photochemical products were compared to literature spectra for identification. Several wavelengths were chosen for photoexcitation, ranging from the S(0)-S(1) origin transition (266.79 nm) to 193 nm. Comparison of the product spectral intensities as a function of photoexcitation wavelength provides information on the wavelength dependence of the product yields. Photoexcitation at 266.79 nm yielded five products (benzyl radical, benzylallenyl radical, 1-phenyl-1,3-butadiene, 1,2-dihydronaphthalene, and naphthalene), with naphthalene and benzylallenyl radicals dominant. At 193 nm, the benzylallenyl radical signal was greatly reduced in intensity, while three additional C(10)H(8) isomeric products were observed. An extensive set of calculations of key stationary points on the ground state C(10)H(10) and C(10)H(9) potential energy surfaces were carried out at the DFT B3LYP/6-311G(d,p) level of theory. Mechanisms for formation of the observed products are proposed based on these potential energy surfaces, constrained by the results of cursory studies of the photochemistry of 1-phenyl-1,3-butadiene and 4-phenyl-1-butyne. A role for tunneling on the excited state surface in the formation of naphthalene is suggested by studies of partially deuterated benzylallene, which blocked naphthalene formation.