Femtosecond Coherence Spectroscopy Research Articles

Theoretical model of femtosecond coherence spectroscopy of vibronic excitons in molecular aggregates.

When used as pump pulses in transient absorption spectroscopy measurements, femtosecond laser pulses can produce oscillatory signals known as quantum beats. The quantum beats arise from coherent superpositions of the states of the sample and are best studied in the Fourier domain using Femtosecond Coherence Spectroscopy (FCS), which consists of one-dimensional amplitude and phase plots of a specified oscillation frequency as a function of the detection frequency. Prior works have shown ubiquitous amplitude nodes and π phase shifts in FCS from excited-state vibrational wavepackets in monomer samples. However, the FCS arising from vibronic-exciton states in molecular aggregates have not been studied theoretically. Here, we use a model of vibronic-exciton states in molecular dimers based on displaced harmonic oscillators to simulate FCS for dimers in two important cases. Simulations reveal distinct spectral signatures of excited-state vibronic-exciton coherences in molecular dimers that may be used to distinguish them from monomer vibrational coherences. A salient result is that, for certain relative orientations of the transition dipoles, the key resonance condition between the electronic coupling and the frequency of the vibrational mode may yield strong enhancement of the quantum-beat amplitude and, perhaps, also cause a significant decrease of the oscillation frequency to a value far lower than the vibrational frequency. Future studies using these results will lead to new insights into the excited-state coherences generated in photosynthetic pigment-protein complexes.

Elementary Electron and Ion Dynamics in Ionized Liquid Water

The ionization of liquid water functions as the principal trigger for a myriad of phenomena that are relevant to radiation chemistry and biology. The earliest events that follow the ionization of water, however, remain relatively unknown. Here, femtosecond coherence spectroscopy is combined with polarization anisotropy measurements to elucidate the ultrafast electron and ion dynamics in ionized water. The results show that strong-field ionization of liquid water produces an aligned p electron distribution. Furthermore, oscillations observed in the polarization anisotropy are suggestive of valence electron motion in the highly reactive H2O+ radical cation, whose lifetime with respect to proton transfer is found to be 196 ± 5 fs. Coherent intermolecular motions that signal initial solvent reorganization and subsequent long-lived ballistic proton transport that involves the H3O+ end product are also detected in the time domain. These results offer new insight into the elementary dynamics of ionized liquid water.

Low frequency dynamics of Cystathionine beta-synthase

Femtosecond coherence spectroscopy is used to study the low frequency dynamics of cystathionine beta-synthase (CBS). CBS is a pyridoxal-5′-phosphate-dependent heme enzyme with cysteine and histidine axial ligands that catalyzes the condensation of serine and homocysteine to form cystathionine. Resonance excitation near the maximum of the ferric state Soret band reveals a mode near ∼40 cm−1 (phase ∼pi/2). The phase indicates that the initial non-equilibrium coherent wavepacket for this mode is dominated by a momentum displacement. This is consistent with doming of the ferric five-coordinate species and suggests photolysis of the histidine ligand. When exciting on the red side of the Soret band, a mode near ∼25 cm−1 is observed that exhibits a phase jump of ∼pi for blue-side excitation. This mode may involve the response of an unphotolyzed fraction of hot ferric six-coordinate species, subsequent to ultrafast non-radiative decay. A strong correlation between the “detuned” coherence spectrum (which reveals higher frequencies) and the Raman spectrum is also demonstrated. Normal coordinate structural decomposition of the ferric heme crystal structure predicts strong saddling, doming, and ruffling modes and they are observed in the coherence spectra. The relative intensities of these modes are monitored as a function of pH in order to explore the potential correlation between redox equilibria, pH, and protein-induced heme structural perturbations. The low frequency spectra of ferrous CBS and its NO-bound complex were also obtained, along with the CO rebinding kinetics. The geminate rebinding of CO to CBS was found to be unusually fast and similar to that of CooA.

Low-Frequency Mode Activity of Heme: Femtosecond Coherence Spectroscopy of Iron Porphine Halides and Nitrophorin

The low-frequency mode activity of metalloporphyrins has been studied for iron porphine-halides (Fe(P)(X), X = Cl, Br) and nitrophorin 4 (NP4) using femtosecond coherence spectroscopy (FCS) in combination with polarized resonance Raman spectroscopy and density functional theory (DFT). It is confirmed that the mode symmetry selection rules for FCS are the same as for Raman scattering and that both Franck-Condon and Jahn-Teller mode activities are observed for Fe(P)(X) under Soret resonance conditions. The DFT-calculated low-frequency (20-400 cm (-1)) modes, and their frequency shifts upon halide substitution, are in good agreement with experimental Raman and coherence data, so that mode assignments can be made. The doming mode is located at approximately 80 cm (-1) for Fe(P)(Cl) and at approximately 60 cm (-1) for Fe(P)(Br). NP4 is also studied with coherence techniques, and the NO-bound species of ferric and ferrous NP4 display a mode at approximately 30-40 cm (-1) that is associated with transient heme doming motion following NO photolysis. The coherence spectra of three ferric derivatives of NP4 with different degrees of heme ruffling distortion are also investigated. We find a mode at approximately 60 cm (-1) whose relative intensity in the coherence spectra depends quadratically on the magnitude of the ruffling distortion. To quantitatively account for this correlation, a new "distortion-induced" Raman enhancement mechanism is presented. This mechanism is unique to low-frequency "soft modes" of the molecular framework that can be distorted by environmental forces. These results demonstrate the potential of FCS as a sensitive probe of dynamic and functionally important nonplanar heme vibrational excitations that are induced by the protein environmental forces or by the chemical reactions in the aqueous phase.

Coherence Spectroscopy Investigations of the Low-Frequency Vibrations of Heme: Effects of Protein-Specific Perturbations

Femtosecond coherence spectroscopy is used to probe the low-frequency (20-200 cm(-1)) vibrational modes of heme proteins in solution. Horseradish peroxidase (HRP), myoglobin (Mb), and Campylobacter jejuni globin (Cgb) are compared and significant differences in the coherence spectra are revealed. It is concluded that hydrogen bonding and ligand charge do not strongly affect the low-frequency coherence spectra and that protein-specific deformations of the heme group lower its symmetry and control the relative spectral intensities. Such deformations potentially provide a means for proteins to tune heme reaction coordinates, so that they can perform a broad array of specific functions. Native HRP displays complex spectral behavior above approximately 50 cm(-1) and very weak activity below approximately 50 cm(-1). Binding of the substrate analog, benzhydroxamic acid, leads to distinct changes in the coherence and Raman spectra of HRP that are consistent with the stabilization of a heme water ligand. The CN derivatives of the three proteins are studied to make comparisons under conditions of uniform heme coordination and spin-state. MbCN is dominated by a doming mode near 40 cm(-1), while HRPCN displays a strong oscillation at higher frequency (96 cm(-1)) that can be correlated with the saddling distortion observed in the X-ray structure. In contrast, CgbCN displays low-frequency coherence spectra that contain strong modes near 30 and 80 cm(-1), probably associated with a combination of heme doming and ruffling. HRPNO displays a strong doming mode near 40 cm(-1) that is activated by photolysis. The damping of the coherent motions is significantly reduced when the heme is shielded from solvent fluctuations by the protein material and reduced still further when T approximately < 50 K, as pure dephasing processes due to the protein-solvent phonon bath are frozen out.

Investigations of Vibrational Coherence in the Low-Frequency Region of Ferric Heme Proteins

Femtosecond coherence spectroscopy is applied to a series of ferric heme protein samples. The low-frequency vibrational spectra that are revealed show dominant oscillations near 40 cm −1. MbCN is taken as a typical example of a histidine-ligated, six-coordinate, ferric heme and a comprehensive spectroscopic analysis is carried out. The results of this analysis reveal a new heme photoproduct species, absorbing near 418 nm, which is consistent with the photolysis of the His 93 axial ligand. The photoproduct undergoes subsequent rebinding/recovery with a time constant of ∼4 ps. The photoproduct lineshapes are consistent with a photolysis quantum yield of 75–100%, although the observation of a relatively strong six-coordinate heme coherence near 252 cm −1 (assigned to ν 9 in the MbCN Raman spectrum) suggests that the 75% lower limit is much more likely. The phase and amplitude excitation profiles of the low-frequency mode at 40 cm −1 suggest that this mode is strongly coupled to the MbCN photoproduct species and it is assigned to the doming mode of the transient penta-coordinated material. The absolute phase of the 40 cm −1 mode is found to be π/2 on the red side of 418 nm and it jumps to 3 π/2 as excitation is tuned to the blue side of 418 nm. The absolute phase of the 40 cm −1 signal is not explained by the standard theory for resonant impulsive stimulated Raman scattering. New mechanisms that give a dominant momentum impulse to the resonant wavepacket, rather than a coordinate displacement, are discussed. The possibilities of heme iron atom recoil after photolysis, as well as ultrafast nonradiative decay, are explored as potential ways to generate the strong momentum impulse needed to understand the phase properties of the 40 cm −1 mode.

Low Frequency Spectral Density of Ferrous Heme: Perturbations Induced by Axial Ligation and Protein Insertion

Femtosecond coherence spectroscopy is used to probe low frequency (20–400 cm −1) modes of the ferrous heme group in solution, with and without 2-methyl imidazole (2MeIm) as an axial ligand. The results are compared to heme proteins (CPO, P450 cam, HRP, Mb) where insertion of the heme into the protein results in redistribution of the low frequency spectral density and in (∼60%) longer damping times for the coherent signals. The major effect of imidazole ligation to the ferrous heme is the “softening” of the low frequency force constants by a factor of ∼0.6 ± 0.1. The functional consequences of imidazole ligation are assessed and it is found that the enthalpic CO rebinding barrier is increased significantly when imidazole is bound. The force constant softening analysis, combined with the kinetics results, indicates that the iron is displaced by only ∼0.2 Å from the heme plane in the absence of the imidazole ligand, whereas it is displaced by ∼0.4 Å when imidazole (histidine) is present. This suggests that binding of imidazole (histidine) as an axial ligand, and the concomitant softening of the force constants, leads to an anharmonic distortion of the heme group that has significant functional consequences.

Low Frequency Modes in Heme Proteins

Abstract The technique of femtosecond coherence spectroscopy is applied to heme protein samples. Strong oscillations are detected near 40 cm-1 in all of the samples studied. Additional modes near 80, 120, and 160 cm-1 are observed in the photochemically active samples. A simple harmonic model is not able to account for the observed relative intensities of these modes or the carrier wavelength dependence of their frequency and phase. As a result, we develop an anharmonic model where the oscillatory signal is damped as the result of heterogeneity in the potential surface. The source of the heterogeneity in the anharmonic potential surface is correlated with the inhomogeneous broadening of the Soret band. The higher harmonics in the photochemically active samples demonstrates that the anharmonic mode is strongly coupled to the ligand photodissociation reaction (i.e., upon photolysis it is displaced far from equilibrium). Moreover, the observation of the ∼40 cm-1 oscillations in all of the iron based heme protein samples, including porphine and protoporphyrin IX model compounds, suggests that this mode is associated with nuclear motion of the core of the porphyrin macrocycle. As a result, we suggest that the reaction coupled oscillations at ∼40 cm-1 and ∼80 cm-1 are a direct reflection of anharmonic heme doming dynamics.

Investigations of Anharmonic Low-Frequency Oscillations in Heme Proteins

The technique of femtosecond coherence spectroscopy is applied to a variety of photostable and photochemically active heme protein samples. With the exception of cobalt-substituted myoglobin, strong oscillations are detected near 40 cm-1 in all of the samples studied. Additional modes near 80, 120, and 160 cm-1 are observed in the photochemically active samples. The amplitude and phase behavior of the low-frequency modes are studied by tuning the pump/probe carrier wavelength across the Soret absorption spectrum. A simple harmonic model is not able to account for the observed relative intensities of these modes or the carrier wavelength dependence of their frequency and phase. As a result, we develop an anharmonic model where the oscillatory signal is damped as the result of heterogeneity in the potential surface. The underlying source of the heterogeneity in the anharmonic potential surface is found to be correlated with the inhomogeneous broadening of the Soret band. The presence of the higher harmonics...

Myoglobin dynamics: evidence for a hybrid solid/fluid state of matter

The dynamics of carboxy-myoglobin (MbCO) in water are studied from photodissociation of the ligand to bimolecular recombination over 12 decades in time to fully characterize the protein functions. Heterodyne-detected transient grating spectroscopy is used to resolve the dynamics of the protein from 10 ns to a few ms and provides a direct observation of the ligand escape. The process is well described by a bi-exponential function with decay rates of 50 and 725 ns at 20°C, suggesting that ligand escape occurs via a well defined pathway. Transient absorption in the Q-band (550–630 nm) also reveals the sensitivity of the electronic transition to the ligand motions. The dynamic range is extended by 10 6 through femtosecond coherence spectroscopy with 7 fs pulses to enable the observation of vibrational modes of energies >1500 cm −1. The power spectrum is calculated by singular value decomposition and vibrational modes involved in the photodissociation are directly observed. The picture that is emerging is that proteins couple solid-like domains to fluid regions to facilitate functions and transport of ligands in and out of the protein to the active site.

Femtosecond coherence spectroscopy using spectrally selective differential photodetection

We present a novel, spectrally selective, detection scheme for pump–probe femtosecond coherence spectroscopy (FCS) that attenuates the non-oscillatory background signal so that the coherent oscillations are more clearly revealed. The method is based on spectral dispersion of the probe beam after it has traversed the sample, followed by detection of spectrally specific portions with a differential detector containing two balanced photodiodes. By appropriately choosing the spectral windows detected by each photodiode, we show how vibrational oscillations can be selectively enhanced or suppressed. Information about the phase behavior of the vibrational modes is also revealed using this detection scheme.

Investigations of amplitude and phase excitation profiles in femtosecond coherence spectroscopy

We present an effective linear response approach to pump–probe femtosecond coherence spectroscopy in the well-separated pulse limit. The treatment presented here is based on a displaced and squeezed state representation for the nonstationary states induced by an ultrashort pump laser pulse or a chemical reaction. The subsequent response of the system to a delayed probe pulse is modeled using closed form nonstationary linear response functions, valid for a multimode vibronically coupled system at arbitrary temperature. When pump–probe signals are simulated using the linear response functions, with the mean nuclear positions and momenta obtained from a rigorous moment analysis of the pump induced (doorway) state, the signals are found to be in excellent agreement with the conventional third-order response approach. The key advantages offered by the moment analysis-based linear response approach include a clear physical interpretation of the amplitude and phase of oscillatory pump–probe signals, a dramatic improvement in computation times, a direct connection between pump–probe signals and equilibrium absorption and dispersion lineshapes, and the ability to incorporate coherence associated with rapid nonradiative surface crossing. We demonstrate these aspects using numerical simulations, and also apply the present approach to the interpretation of experimental amplitude and phase measurements on reactive and nonreactive samples of the heme protein myoglobin. The role played by inhomogeneous broadening in the observed amplitude and phase profiles is discussed in detail. We also investigate overtone signals in the context of reaction driven coherent motion.

Excited State Coherent Vibrational Motion in Deoxymyoglobin

Femtosecond coherence spectroscopy of deoxymyoglobin has been performed under excitation in the lowest electronic transition (Q band) and probing in the Soret band. Under these excitation conditions, ground state low-frequency vibrational motions are strongly suppressed with respect to Soret excitation. Evidence is presented for a contribution of excited state vibrational dynamics to the coherent response.

Femtosecond Multicolor Pump−Probe Spectroscopy of Ferrous Cytochrome c

Ligand photolysis and subsequent electronic and structural relaxation, followed by ligand recombination, in ferrous cytochrome c (cyt c) have been studied using ultrafast laser spectroscopy. A broad-band white-light continuum, generated by amplified pulses from a Ti:sapphire laser, was used to monitor the transient absorption spectra of cyt c in the Soret and Q bands following 50-fs pulsed photoexcitation at 400 nm. The reconstructed photoproduct absorption spectrum is found to closely resemble that of a model pentacoordinate histidine-ligated complex, microperoxidase (MP-8), suggesting methionine photolysis. Vibrational modes at ∼40, ∼80, and ∼220 cm-1 are observed in femtosecond coherence spectroscopy (FCS) measurements, which also indicates photodissociation of the methionine ligand. The quantum yield of ligand photolysis is found to be ≥80%, which is consistent with the ultrafast photolysis time constant (≤40 fs) needed to induce coherent oscillations in the FCS measurements. The combination of high quantum yield and short time constant helps to resolve the longstanding question of the origin of the short lifetime (τe) and large Soret-state electronic damping factor (Γe) previously found in cytochrome c. We propose a simple multilevel model to describe the observed experimental data. The global analysis of the measured kinetics leads to a characterization of the major kinetic rates, including the 6.2-ps geminate rebinding of methionine to the heme iron.

Investigations of Coherent Vibrational Oscillations in Myoglobin

The technique of femtosecond coherence spectroscopy (FCS) is applied to the heme protein myoglobin. Photostable samples of deoxy myoglobin (Mb) and photochemically active samples of the nitric oxide adduct (MbNO) are investigated. The pump-induced change in the probe transmittance for both samples displays coherent oscillations that, when transformed into the frequency domain, are in agreement with the resonance Raman spectrum of deoxy Mb. This indicates that the coherences associated with the photoreactive sample (MbNO) arise from the rapidly changing forces appearing in the crossing region(s) between the reactant and product state potential energy surfaces. The relative phase and amplitude of the Fe−His vibration, associated with the sole covalent linkage between the heme and the protein, are analyzed as a function of sample state and pump/probe carrier frequency. The dependence of the phase on carrier frequency is found to be significantly different for the “field driven” coherence in Mb and the “reaction driven” coherence in MbNO. In MbNO we observe a dip in amplitude and a phase flip near 439 nm for the Fe−His mode, whereas in deoxy Mb we observe a nearly constant phase and amplitude for this mode across the Soret absorption band. These observations are shown to be in good agreement with a simple theoretical model of the pump−probe experiment. Finally, we present recent observations of strong low-frequency oscillations, occurring near 40 cm-1 in both species and near 80 cm-1 for MbNO.

Application of femtosecond coherence spectroscopy to the observation of nuclear motions in heme proteins and transparent solutions

The technique of femtosecond coherence spectroscopy (FCS) was applied to simple transparent organic fluids (chloroform and fenchone) and light-absorbing heme proteins (myoglobin derivatives). This study demonstrates that time-domain coherence measurements are in good agreement with standard frequency-domain Raman and resonance Raman scattering experiments. The unique capability of FCS to detect the low-wavenumber nuclear motions driven by the pumping fields and by the electronic state changes associated with photoinduced chemical reactions is also demonstrated. The latter response cannot be probed using traditional Raman spectroscopy. In all studies of myoglobin derivatives a low-wavenumber mode was observed near 40 cm−1. A sequence of low-wavenumber modes was also observed near 80, 120 and 160 cm−1 that could be an indication of an overtone sequence. Copyright © 2000 John Wiley & Sons, Ltd.

Low frequency vibrational modes in proteins: changes induced by point-mutations in the protein-cofactor matrix of bacterial reaction centers.

As a step toward understanding their functional role, the low frequency vibrational motions (<300 cm-1) that are coupled to optical excitation of the primary donor bacteriochlorophyll cofactors in the reaction center from Rhodobacter sphaeroides were investigated. The pattern of hydrogen-bonding interaction between these bacteriochlorophylls and the surrounding protein was altered in several ways by mutation of single amino acids. The spectrum of low frequency vibrational modes identified by femtosecond coherence spectroscopy varied strongly between the different reaction center complexes, including between different mutants where the pattern of hydrogen bonds was the same. It is argued that these variations are primarily due to changes in the nature of the individual modes, rather than to changes in the charge distribution in the electronic states involved in the optical excitation. Pronounced effects of point mutations on the low frequency vibrational modes active in a protein-cofactor system have not been reported previously. The changes in frequency observed indicate a strong involvement of the protein in these nuclear motions and demonstrate that the protein matrix can increase or decrease the fluctuations of the cofactor along specific directions.

Femtosecond coherence spectroscopy of heme proteins

Femtosecond pump-probe experiments are used to study the photophysics and photochemistry of heme proteins on ultrafast time scales. Using electronic relaxation measurements combined with reaction-driven coherence signals, we determine that, after the photoexcitation of myoglobin (Mb)NO, the bond-breaking time is on the order of the electronic dephasing time (∼ 20 fs) and the electronic ground state of deoxy Mb appears on a time scale of 80 ± 30 fs. This latter time scale is linked to the Fe-His bond compression (TFe-His/2 ∼ 75 fs), which is initiated by the photolytic depletion of electron density from the antibonding dz2 orbital. The subsequent depression of the dx2-y2 orbital energy, and its population as the iron moves out of plane, leads to the formation of the S = 2 electronic ground state and drives the doming of the heme (Tdome/4 ∼ 100 fs). Thus, the initial stage of heme doming is completed in TFe-His/2 + Tdome/4 ∼ 180 fs. Samples of hemoglobin (Hb)NO display a strong coherent Fe-His stretching vibration at 230 cm−1, which appears immediately (∼ 200 fs) after photodissociation. The data also show a lower-frequency component at 95 cm−1, which we assign to heme doming. The reaction driven coherence of HbNO is observed to be much stronger than the impulsively stimulated Raman coherence of Hb, as might be expected, based on the large relative displacement between reactant and product equilibrium position. The Raman coherence of deoxy Mb is also studied as a function of temperature. As the temperature decreases from 290 to 10 K, the amplitude of the 370 cm−1 mode increases while the amplitude of the 220 cm−1 Fe-His stretching mode decreases. Finally, we report preliminary observations of the first Raman coherences in samples of another heme protein, cytochrome P450. © 1996 John Wiley & Sons, Inc.

Femtosecond coherence spectroscopy of heme proteins

Femtosecond coherence spectroscopy of heme proteins