Vibrational Relaxation in beta-Carotene Probed by Picosecond Stokes and Anti-Stokes Resonance Raman Spectroscopy

Picosecond time-resolved Stokes and anti-Stokes resonance Raman spectra of all-trans-beta-carotene are obtained and analyzed to reveal the dynamics of excited-state (S(1)) population and decay, as well as ground-state vibrational relaxation. Time-resolved Stokes spectra show that the ground state recovers with a 12.6 ps time constant, in agreement with the observed decay of the unique S(1) Stokes bands. The anti-Stokes spectra exhibit no peaks attributable to the S(1) (2A(g) (-)) state, indicating that vibrational relaxation in S(1) must be nearly complete within 2 ps. After photoexcitation there is a large increase in anti-Stokes scattering from ground-state modes that are vibrationally excited through internal conversion. The anti-Stokes data are fit to a kinetic scheme in which the C=C mode relaxes in 0.7 ps, the C-C mode relaxes in 5.4 ps and the C-CH(3) mode relaxes in 12.1 ps. These results are consistent with a model for S(1)-S(0) internal conversion in which the C=C mode is the primary acceptor, the C-C mode is a minor acceptor, and the C-CH(3) mode is excited via intramolecular vibrational energy redistribution.     

State preparation and excited electronic and vibrational behavior in hemes

The temporally overlapping, ultrafast electronic and vibrational dynamics of a model five-coordinate, high-spin heme in a nominally isotropic solvent environment has been studied for the first time with three complementary ultrafast techniques: transient absorption, time-resolved resonance Raman Stokes, and time-resolved resonance Raman anti-Stokes spectroscopies. Vibrational dynamics associated with an evolving ground-state species dominate the observations. Excitation into the blue side of the Soret band led to very rapid S2 --> S1 decay (sub-100 fs), followed by somewhat slower (800 fs) S1 --> S0 nonradiative decay. The initial vibrationally excited, non-Boltzmann S0 state was modeled as shifted to lower energy by 300 cm(-1) and broadened by 20%. On a approximately 10 ps time scale, the S0 state evolved into its room-temperature, thermal distribution S0 profile largely through VER. Anti-Stokes signals disappear very rapidly, indicating that the vibrational energy redistributes internally in about 1-3 ps from the initial accepting modes associated with S1 --> S0 internal conversion to the rest of the macrocycle. Comparisons of anti-Stokes mode intensities and lifetimes from TRARRS studies in which the initial excited state was prepared by ligand photolysis [Mizutani, T.; Kitagawa, T. Science 1997, 278, 443, and Chem. Rec. 2001, 1, 258] suggest that, while transient absorption studies appear to be relatively insensitive to initial preparation of the electronic excited state, the subsequent vibrational dynamics are not. Direct, time-resolved evaluation of vibrational lifetimes provides insight into fast internal conversion in hemes and the pathways of subsequent vibrational energy flow in the ground state. The overall similarity of the model heme electronic dynamics to those of biological systems may be a sign that the protein's influence upon the dynamics of the heme active site is rather subtle.     

Resonance Raman Structural Evidence that the Cis-to-Trans Isomerization in Rhodopsin Occurs in Femtoseconds

Picosecond time-resolved resonance Raman spectroscopy is used to probe the structural changes of rhodopsin's retinal chromophore as the cis-to-trans isomerization reaction occurs that initiates vision. Room-temperature resonance Raman spectra of rhodopsin's photoproduct with time delays from -0.7 to 20.8 ps are measured using 2.2 ps, 480 nm pump and 1.5 ps, 600 nm probe pulses. Hydrogen-out-of-plane (HOOP) modes at 852, 871, and 919 cm(-1), fingerprint peaks at 1272, 1236, 1211, and 1166 cm(-1), and a broad red-shifted ethylenic band at 1530 cm(-1) are present at the earliest positive pump-probe time delay of 0.8 ps, indicating that the chromophore is already in a strained, all-trans configuration. Kinetic analyses of both the HOOP and ethylenic regions of the photoproduct spectra reveal that these features grow in with fast ( approximately 200 fs) and slow ( approximately 2-3 ps) components. These data provide the first structural evidence that photorhodopsin has a thermally unrelaxed, torsionally strained all-trans chromophore within approximately 1 ps, and possibly within 200 fs, of photon absorption. Following this ultrafast product formation, the all-trans chromophore cools and conformationally relaxes within a few picoseconds to form bathorhodopsin. This cooling process is revealed as an ethylenic frequency blue-shift of 6 cm(-1) (tau approximately 3.5 ps) as well as an ethylenic width narrowing (tau approximately 2 ps). The ultrafast production of photorhodopsin is likely accompanied by an impulsively driven, localized protein response. More delocalized protein modes are unable to relax on this ultrafast time scale enabling the chromophore-protein complex to store the large amounts of photon energy (30-35 kcal/mol) that are subsequently used to drive activating protein conformational changes.     

Development of simultaneous frequency- and time-resolved coherent anti-Stokes Raman scattering for ultrafast detection of molecular Raman spectra

The development of a time-resolved coherent anti-Stokes Raman scattering (CARS) variant for use as a probe of excited electronic state Raman-active modes following excitation with an ultrafast pump pulse is detailed. Application of this technique involves a combination of broadband fs-time scale pulses and a narrowband pulse of ps duration that allows multiplexed detection of the CARS signal, permitting direct observation of molecular Raman frequencies and intensities with time resolution dictated by the broadband pulses. Thus, this nonlinear optical probe, designated fs/ps CARS, is suitable for observation of Raman spectral evolution following excitation with a pump pulse. Because of the spatial separation of the CARS output signal relative to the three input beams inherent in a folded BOXCARS arrangement, this technique is particularly amenable to probing low-frequency vibrational modes, which play a significant role in accepting vibrational energy during intramolecular vibrational energy redistribution within electronically excited states. Additionally, this spatial separation allows discrimination against strong fluorescence signal, as demonstrated in the case of rhodamine 6G.     

Second-chance forward isomerization dynamics of the red/green cyanobacteriochrome NpR6012g4 from Nostoc punctiforme

The primary ultrafast Z-to-E isomerization photodynamics of the phytochrome-related cyanobacteriochrome NpR6012g4 from Nostoc punctiforme was studied by transient absorption pump-dump-probe spectroscopy. A 2 ps dump pulse resonant with the stimulated emission band depleted 21% of the excited-state population, while the initial photoproduct Lumi-R was depleted by only 11%. We observed a red-shifted ground-state intermediate (GSI) that we assign to a metastable state that failed to isomerize fully. Multicomponent global analysis implicates the generation of additional Lumi-R from the GSI via crossing over the ground-state thermal barrier for full isomerization, explaining the discrepancy between excited-state and Lumi-R depletion by the dump pulse. This second-chance ground-state dynamics provides a plausible explanation for the unusually high quantum yield of 40% for the primary isomerization step in the forward reaction of NpR6012g4.     

Ultrafast dynamics of myoglobin probed by time-resolved resonance Raman spectroscopy

Recent experimental work carried out in this laboratory on the ultrafast dynamics of myoglobin (Mb) is summarized with a stress on structural and vibrational energy relaxation. Studies on the structural relaxation of Mb following CO photolysis revealed that the structural change of heme itself, caused by CO photodissociation, is completed within the instrumental response time of the time-resolved resonance Raman apparatus used (approximately 2 ps). In contrast, changes in the intensity and frequency of the iron-histidine (Fe-His) stretching mode upon dissociation of the trans ligand were found to occur in the picosecond regime. The Fe-His band is absent for the CO-bound form, and its appearance upon photodissociation was not instantaneous, in contrast with that observed in the vibrational modes of heme, suggesting appreciable time evolution of the Fe displacement from the heme plane. The band position of the Fe-His stretching mode changed with a time constant of about 100 ps, indicating that tertiary structural changes of the protein occurred in a 100-ps range. Temporal changes of the anti-Stokes Raman intensity of the v4 and v7 bands demonstrated immediate generation of vibrationally excited heme upon the photodissociation and decay of the excited populations, whose time constants were 1.1 +/- 0.6 and 1.9 +/- 0.6 ps, respectively. In addition, the development of the time-resolved resonance Raman apparatus and prospects in this research field are described.     

Product formation in rhodopsin by fast hydrogen motions

The photochemical cis-trans isomerization of retinal in rhodopsin is investigated by structure sampling and excited state QM/MM trajectories with surface hopping. The calculations uncover the motions responsible for photoproduct formation and elucidate the reasons behind the efficient photoisomerization in the primary event of visual transduction.     

CARS-spectra of excited states of metalloporphyrins

It is shown that resonance coherent anti-Stokes Raman scattering (RCARS) spectra of Cu- and Ni-octaethylporphyrins contain lines corresponding to molecule vibrations both in the ground and excited electronic states. Mechanisms are given which lead to the appearance of vibrations in the excited state in RCARS spectra. The RCARS methods are shown to be promising for comparing molecule vibrations in different electronic states.     

Characterizing excited states of CH5+ with diffusion Monte Carlo

The spectroscopy and dynamics of protonated methane have been of long-standing interest due to the unusual and highly fluxional behavior of CH5+. This reflects the fact that the ground-state wave function for CH5+ has nearly equal amplitude at the 120 equivalent minima and at the saddle points that connect these minima. While low-resolution spectra of CH5+ have been assigned, the nature of the couplings between the CH stretches and the low-frequency modes is not as well characterized. An understanding of this will be important in the interpretation of rotationally resolved spectra. In this work, fixed-node diffusion Monte Carlo techniques are used to calculate energies and probability amplitudes for several excited states. The calculated energies are shown to be in good agreement with previously reported vibrational configuration interactions calculations. Analysis of the 12-dimensional probability amplitudes shows that there are strong couplings between the high-frequency CH stretch and HCH bend motions and the low-frequency modes that lead to isomerization CH5+.     

斯托克斯和反斯托克斯拉曼位移差与时间反演对称性

The anti-Stokes scattering was defined as the corresponding time-reversed process of the Stokes scattering. Based on the time-reversal symmetry the differences between one Stokes and its corresponding anti Stokes Raman shifts have been theoretically demonstrated for the systems with Kramers-degenerate ground or final states. In the former cases the Stokes and its corresponding anti-Stokes Raman lines should display on the same side of the excited line. In the latter cases, they show major differences on two sides of the excited line. However, the whole normal Stokes Raman spectrum of a system may be still symmetric to the whole anti-Stokes spectrum, except in the cases of resonant, or laser, or circularly polarized excitation Raman scattering. This is a preliminary theoretical predication which needs more experimental and theoretical investigations.     

Vibrational coherence spectroscopy of the heme domain in the CO-sensing transcriptional activator CooA

Femtosecond vibrational coherence spectroscopy was used to investigate the low-frequency vibrational dynamics of the heme in the carbon monoxide oxidation activator protein (CooA) from the thermophilic anaerobic bacterium Carboxydothermus hydrogenoformans (Ch-CooA). Low frequency vibrational modes are important because they are excited by the ambient thermal bath (k(B)T = 200 cm(-1)) and participate in thermally activated barrier crossing events. However, such modes are nearly impossible to detect in the aqueous phase using traditional spectroscopic methods. Here, we present the low frequency coherence spectra of the ferric, ferrous, and CO-bound forms of Ch-CooA in order to compare the protein-induced heme distortions in its active and inactive states. Distortions take place predominantly along the coordinates of low-frequency modes because of their weak force constants, and such distortions are reflected in the intensity of the vibrational coherence signals. A strong mode near ~90 cm(-1) in the ferrous form of Ch-CooA is suggested to contain a large component of heme ruffling, consistent with the imidazole-bound ferrous heme crystal structure, which shows a significant protein-induced heme distortion along this coordinate. A mode observed at ~228 cm(-1) in the six-coordinate ferrous state is proposed to be the ν(Fe-His) stretching vibration. The observation of the Fe-His mode indicates that photolysis of the N-terminal α-amino axial ligand takes place. This is followed by a rapid (~8.5 ps) transient absorption recovery, analogous to methionine rebinding in photolyzed ferrous cytochrome c. We have also studied CO photolysis in CooA, which revealed very strong photoproduct state coherent oscillations. The observation of heme-CO photoproduct oscillations is unusual because most other heme systems have CO rebinding kinetics that are too slow to make the measurement possible. The low frequency coherence spectrum of the CO-bound form of Ch-CooA shows a strong vibration at ~230 cm(-1) that is broadened and up-shifted compared to the ν(Fe-His) of Rr-CooA (216 cm(-1)). We propose that the stronger Fe-His bond is related to the enhanced thermal stability of Ch-CooA and that there is a smaller (time dependent) tilt of the histidine ring with respect to the heme plane in Ch-CooA. The appearance of strong modes at ~48 cm(-1) in both the ferrous and CO-bound forms of Ch-CooA is consistent with coupling of the heme doming distortion to the photolysis reaction in both samples. Upon CO binding and protein activation, a heme mode near 112 ± 5 cm(-1) disappears, probably indicating a decreased heme saddling distortion. This reflects changes in the heme environment and geometry that must be associated with the conformational transition activating the DNA-binding domain. Protein-specific DNA binding to the CO-bound form of Ch-CooA was also investigated, and although the CO rebinding kinetics are significantly perturbed, there are negligible changes in the low-frequency vibrational spectrum of the heme.     

Femtosecond stimulated Raman study of excited-state evolution in bacteriorhodopsin

Femtosecond time-resolved stimulated Raman spectroscopy (FSRS) is used to examine the photoisomerization dynamics in the excited state of bacteriorhodopsin. Near-IR stimulated emission is observed in the FSRS probe window that decays with a 400-600-fs time constant. Additionally, dispersive vibrational lines appear at the locations of the ground-state vibrational frequencies and decay with a 260-fs time constant. The dispersive line shapes are caused by a nonlinear effect we term Raman initiated by nonlinear emission (RINE) that generates vibrational coherence on the ground-state surface. Theoretical expressions for the RINE line shapes are developed and used to fit the spectral and temporal evolution of the spectra. The rapid 260-fs decay of the RINE peak intensity, compared to the slower evolution of the stimulated emission, indicates that the excited-state population moves in approximately 260 fs to a region on the potential energy surface where the RINE signal is attenuated. This loss of RINE signal is best explained by structural evolution of the excited-state population along multiple low-frequency modes that carry the molecule out of the harmonic photochemically inactive Franck-Condon region and into the photochemically active geometry.     

Stimulated Raman laser excitation of spontaneous resonance Raman scattering

The efficient conversion of the second and third harmonics of a Nd YAG laser to near UV radiation in the 395–500 nm range by stimulated Stokes (and anti-Stokes) Raman scattering (SRS) in a 1 m Raman oscillator containing compressed H₂ or D₂ is used as an excitation source for spontaneous resonance Raman spectroscopy (RRS). SRS excited RR spectra are shown for the anion radical of tetracyanoquinodimethane (TCNQ).     

Ultrafast excited-state dynamics preceding a ligand trans-cis isomerization of fac-[Re(Cl)(CO)3(t-4-styrylpyridine)2] and fac-[Re(t-4-styrylpyridine)(CO)3(2,2'-bipyridine)]+

UV-vis absorption and resonance Raman spectra of the complexes fac-[Re(Cl)(CO)3(stpy)2] and fac-[Re(stpy)(CO)3(bpy)]+ (stpy = t-4-styrylpyridine, bpy = 2,2'-bipyridine) show that their lowest absorption bands are dominated by stpy-localized intraligand (IL) pi pi* transitions. For the latter complex a Re --> bpy transition contributes to the low-energy part of the absorption band. Optical population of the 1IL excited state of fac-[Re(Cl)(CO)3(stpy)2] is followed by an intersystem crossing (< or =0.9 ps) to an 3IL state with the original planar trans geometry of the stpy ligand. This state undergoes a approximately 90 degrees rotation around the stpy C=C bond with a 11 ps time constant. An electronically excited species with an approximately perpendicular orientation of the phenyl and pyridine rings of the stpy ligand is formed. Conversion to the ground state and isomerization occurs in the nanosecond range. Intraligand excited states of fac-[Re(stpy)(CO)3(bpy)]+ show the same behavior. Moreover, it was found that the planar reactive 3IL excited state is rapidly and efficiently populated after optical excitation into the Re --> bpy 1MLCT excited state. A 1MLCT --> 3MLCT intersystem crossing takes place first with a time constant of 0.23 ps followed by an intramolecular energy transfer from the ReI(CO)3(bpy) chromophore to a stpy-localized 3IL state with a 3.5 ps time constant. The fast rate ensures complete conversion. Coordination of the stpy ligand to the ReI center thus switches the ligand trans-cis isomerization mechanism from singlet to triplet (intramolecular sensitization) and, in the case of fac-[Re(stpy)(CO)3(bpy)]+, opens an indirect pathway for population of the reactive 3IL excited state via MLCT states.     

Photoisomerization of disperse red 1 studied with transient absorption spectroscopy and quantum chemical calculations

The photoisomerization of the push-pull substituted azo dye Disperse Red 1 is studied using femtosecond time-resolved absorption spectroscopy and other spectroscopic and computational techniques. In comparison with azobenzene, the pipi* state is more stabilized by the effects of push-pull substitution than the npi* state, but the latter is probably still the lowest in energy. This conclusion is based on the kinetics, anisotropy of the excited state absorption spectrum, the spectra of the ground states, and quantum chemical calculations. The S(1)(npi*) state is formed from the initially excited pipi* state in <0.2 ps, and decays to the ground state with time constants of 0.9 ps in toluene, 0.5 ps in acetonitrile, and 1.4 ps in ethylene glycol. Thermal isomerization transforms the Z isomer produced to the more stable E isomer with time constants of 29 s (toluene), 28 ms (acetonitrile), and 2.7 ms (ethylene glycol). The pathway of photoisomerization is likely to be rotation about the N=N bond. Quantum chemical calculations indicate that along the inversion pathway ground and excited state energy surfaces remain well separated, whereas rotation leads to a region where conical intersections can occur. For the ground-state Z to E isomerization, conclusive evidence is lacking, but inversion is more probably the favored pathway in the push-pull substituted systems than in the parent azobenzene.     

Photoexcited structural dynamics of an azobenzene analog 4-nitro-4'-dimethylamino-azobenzene from femtosecond stimulated Raman

Azobenzenes are used in many applications because of their robust and reversible light induced trans?cis isomerization about the N=N bond, but the mechanism of this ultrafast reaction has not been conclusively defined. Addressing this problem we have used Femtosecond Stimulated Raman Spectroscopy (FSRS) to determine the structural transients in the trans→cis photoisomerization of the azobenzene derivative, 4-nitro-4'-dimethylamino-azobenzene (NDAB). Key marker modes, such as the 1570/1590 cm(-1) NO(2) stretch and the 1630 cm(-1) C-N(Me)(2) stretch, enable the separation and analysis of distinct trans and cis photoproduct dynamics revealing the 400 fs Frank-Condon relaxation, the 800 fs timescale of the cis product formation and the 2 ps emergence and 8 ps relaxation of the unsuccessful ground state trans species. Based on these observations, we propose a reaction mechanism, including initial dilation of the CNN bend later joined by quick movement along the CCNN, CNNC and NNCC torsional coordinates that constitutes a mixed inversion-rotation mechanism.     

Perturbative theory and modeling of electronic-resonance-enhanced coherent anti-Stokes Raman scattering spectroscopy of nitric oxide

A theory is developed for three-laser electronic-resonance-enhanced (ERE) coherent anti-Stokes Raman scattering (CARS) spectroscopy of nitric oxide (NO). A vibrational Q-branch Raman polarization is excited in the NO molecule by the frequency difference between visible Raman pump and Stokes beams. An ultraviolet probe beam is scattered from the induced Raman polarization to produce an ultraviolet ERE-CARS signal. The frequency of the ultraviolet probe beam is selected to be in electronic resonance with rotational transitions in the A (2)Sigma(+)<--X (2)Pi (1,0) band of NO. This choice results in a resonance between the frequency of the ERE-CARS signal and transitions in the (0,0) band. The theoretical model for ERE-CARS NO spectra has been developed in the perturbative limit. Comparisons to experimental spectra are presented where either the probe laser was scanned with fixed Stokes frequency or the Stokes laser was scanned with fixed probe frequency. At atmospheric pressure and an NO concentration of 100 ppm, good agreement is found between theoretical and experimental spectral peak locations and relative intensities for both types of spectra. Factors relating to saturation in the experiments are discussed, including implications for the theoretical predictions.     

Hybrid four-wave mixing in liquid pyridine

Four-wave mixing spectra for liquid pyridine are obtained with broadband laser beam. A comparison of Stokes and anti-Stokes spectra allows the assignment of several lines (951, 991, 1030 cm^?1) to CARS and CSRS processes. Two additional moderately intense lines on the Stokes side between 951 and 991 cm^?1 as well as 991 and 1030 cm^?1 , which do not appear on the anti-Stokes side, are assigned to a “hybrid four-wave mixing” process, in which two active Raman modes of pyridine are involved. This process previously unrevealed in experiment seems to be important for correct assignment of the spectral lines in four-wave mixing experiments with a broadband laser beam.     

A molecular dynamics study of Lys-Trp-Lys: structure and dynamics in solution following photoexcitation

We report studies of the structure and dynamics of a tripeptide Lys-Trp-Lys (KWK) in aqueous solution following photoexcitation by molecular dynamics simulations. For ground-state KWK, we observe three stable conformations with free energy differences of less than 5.2 kJ/mol. Each conformer is stabilized by a pi-cation interaction between one of three protonated amino groups and the indole moiety. For the excited state of tryptophan in KWK, the simulated molecular dynamics of the three isomers are similar, all in good agreement with recent femtosecond experiments (J. Phys. Chem. B 2005, 109, 16901). Specifically, we observe: (1) the fluorescence anisotropy is dominated by a single-exponential component and decays in approximately 130 ps, (2) the total dynamic Stokes shift reaches approximately 2700 cm(-1), and (3) the excited state relaxation dynamics occurs on several time scales ranging from femtoseconds to tens of picoseconds. The relaxation dynamics involve rapid initial response of neighboring water, followed by local motions of flexible peptide chains. These processes drive global restructuring of the tripeptide on a rather flat energy surface, inducing slower dynamics evident in both the water and protein contributions to the stabilization energy of the photoexcited chromophore. The water and protein dynamics are strongly correlated. On a still longer time scale, we observe isomerization of two excited state conformers to the other most stable one, an analogue for evolution of trajectories along the funnel on the rugged free energy landscape to the final "native" state. Our studies suggest new experiments to detect this unique dynamics.     

ABSORBANCE and CIRCULAR DICHROISM SPECTRA OF 1-C1S PHOTOPRODUCT FORMED BY IRRADIATING FROG RHODOPSIN

Abstract— We showed by spectrophotometry and HPLC that a photoproduct having 7-cis retinal (1-cis photoproduct) can be derived from the photoisomerization of frog lumirhodopsin (L) and metarhodopsin I (M I). The efficiency of the isomerization was higher in M I than in L. The absorption maximum of the 1-cis photoproduct at -20°C is at 455 nm, and its maximum absorbance 1.1 times as large as that of rhodopsin. The photoproduct exhibited two positive CD bands at 450 nm α-band) and 320 nm (β-band); the molecular ellipticity at a-band ([θ] = 73000) being larger than that of rhodopsin ([θ] = 61000). Re-examination of the absorption spectra of rhodopsin intermediates gave the absorption maxima of L. M 1 and M 111 to be 522, 482 and 475 nm, respectively.