Picosecond Time-resolved Resonance Raman Spectroscopy Research Articles

Role of atomic contacts in vibrational energy transfer in myoglobin.

Heme proteins are ideal systems to investigate vibrational energy flow at the atomic level. Upon photoexcitation, a large amount of excess vibrational energy is selectively deposited on heme due to extremely fast internal conversion. This excess energy is redistributed to the surrounding protein moiety and then to water. Vibrational energy flow in myoglobin (Mb) was examined using picosecond time-resolved anti-Stokes ultraviolet resonance Raman (UVRR) spectroscopy. We used the Trp residue directly contacting the heme group as a selective probe for vibrationally excited populations. Trp residues were placed at different position close to the heme by site-directed mutagenesis. This technique allows us to monitor the excess energy on residue-to-residue basis. Anti-Stokes UVRR measurements for Mb mutants suggest that the dominant channel for energy transfer in Mb is the pathway through atomic contacts between heme and nearby amino acid residues as well as that between the protein and solvent water. It is found that energy flow through proteins is analogous to collisional exchange processes in solutions.

Ultrafast protein dynamics of hemoglobin as studied by picosecond time-resolved resonance Raman spectroscopy

Time-resolved resonance Raman spectroscopy on human adult hemoglobin (HbA) following ligand photolysis revealed that the frequency of the iron–histidine stretching [ν(Fe–His)] mode exhibited a 2-cm−1 downshift with a time constant of about 300ps, suggesting a structural change in the heme pocket following the ligand photolysis. Low-frequency heme modes suggested that the primary metastable form of HbA has a more disordered orientation of propionates and a less strained environment than the deoxy form. The latter fact is consistent with the experimental observation that the ν(Fe–His) frequency of the metastable form is higher than the deoxy form. The present study shows that HbA adopts a metastable structure within the instrument response time and remains little changed in the subnanosecond to nanosecond time regime. Characteristics of the primary protein response of HbA based on the comparison of the results of HbA with those of the isolated chains and myoglobin are discussed.

Protein Response to Photoreaction Probed by Picosecond Time-resolved Ultraviolet Resonance Raman Spectroscopy

Time-resolved resonance Raman spectroscopy is able to provide site-selective information on protein dynamics. In this review, we will describe our apparatus of picosecond time-resolved ultraviolet resonance Raman spectroscopy and recent application to protein dynamics of bacteriorhodopsin.

High-Yield Singlet Fission in a Zeaxanthin Aggregate Observed by Picosecond Resonance Raman Spectroscopy

We report high-yield triplet generation by singlet fission upon photoexcitation of a new aggregate of the carotenoid all-trans 3R,3'R-zeaxanthin. The yield is determined by picosecond time-resolved resonance Raman spectroscopy, which allows direct characterization and quantification of triplet excited-state signatures and ground-state depletion. The technique and analysis reveals that triplets form within picoseconds. A quantum yield of 90-200% is derived with the assumption of weak exciton-coupling in the zeaxanthin aggregate.

ChemInform Abstract: Solvent Effects on the ag C=C Stretching Mode in the 21A- g Excited State of β-Carotene and Two Derivatives: Picosecond Time-Resolved Resonance Raman Spectroscopy

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Ultrafast Time-Resolved Transient Absorption and Resonance Raman Spectroscopy Study of the Photodeprotection and Rearrangement Reactions of p-Hydroxyphenacyl Caged Phosphates

The kinetics and mechanism of the photodeprotection and rearrangement reactions for the pHP phototrigger compounds p-hydroxyphenacyl diethyl phosphate (HPDP) and diphenyl phosphate (HPPP) were studied using transient absorption (TA) and picosecond time-resolved resonance Raman (ps-TR(3)) spectroscopy. TA spectroscopy was employed to detect the dynamics of the triplet precursor decay as well as to investigate the influence of the solvent and leaving group on the triplet quenching process. Ps-TR(3) spectroscopy was used to directly monitor the formation dynamics for the photosolvolytic rearrangement product and its solvent and leaving group dependence. The TA and TR(3) spectroscopy experiments were also used to characterize the structural and electronic properties of the triplet precursor to the HPDP and HPPP deprotection reactions. The solvent effect observed in conjunction with the leaving group dependence of the triplet decay dynamics are consistent with a concerted solvent assisted triplet cleavage through a heterolytic mechanism for the HPDP and HPPP photodeprotection process. Correlation of the dynamics between the deprotection and rearrangement processes reveals there is a consecutive mechanism and the involvement of an intermediate between the two reaction steps. The reaction rate of the deprotection and rearrangement steps and the influence of the solvent and leaving group were determined and evaluated based on kinetic modeling of the dynamical data obtained experimentally for HPDP and HPPP in H(2)O/MeCN mixed solvents with varying water concentration in the solvent system. A solvation complex with a contact ion pair character was proposed to be the intermediate involved in the deprotection and rearrangement pathway. The results here combined with our previous study on the photophysical events occurring on the early picosecond time scale (Ma; et al. J. Am. Chem. Soc. 2005, 127, 1463-1472) provide a real time overall mechanistic description for the photodeprotection and rearrangement reactions of pHP caged phosphate phototrigger compounds.

Photoreduction of 4,4‘-Bipyridine by Amines in Acetonitrile−Water Mixtures: Influence of H-Bonding on the Ion-Pair Structure and Dynamics

The photoreduction of 4,4'-bipyridine (44BPY) by diazabicyclo[2.2.2]octane and triethylamine (TEA) is investigated by using picosecond transient absorption and time-resolved resonance Raman spectroscopy in various acetonitrile-water mixtures. The results are interpreted on the basis of a preferential solvation effect resulting from the presence of a specific interaction between 44BPY and water by hydrogen bonding. Below 10% water, the free 44BPY species is dominant and leads upon photoreduction to a contact ion pair that undergoes efficient intrapair proton transfer if TEA is the amine donor. Above 10% water, most of the 44BPY population is H-bonded and leads upon photoreduction to a hydrated ion pair in which the intrapair proton transfer is inhibited. Instead, the 44BPY(-*) species is protonated by water through the hydrogen bond with a rate constant that increases by more than 3 orders of magnitude on going from 10% to 100% water. The dependence of this rate constant on the solvent mixture composition suggests that the reaction of intracomplex proton transfer is controlled by the hydration of the residual OH(-) species by three molecules of water, leading to a trihydrated HO(-)(H(2)O)(3) species.

Structural Characterization of the Proximal and Distal Histidine Environment of Cytoglobin and Neuroglobin

Cytoglobin (Cgb) and neuroglobin (Ngb) are the first examples of hexacoordinated globins from humans and other vertebrates in which a histidine (His) residue at the sixth position of the heme iron is an endogenous ligand in both the ferric and ferrous forms. Static and time-resolved resonance Raman and FT-IR spectroscopic techniques were applied in examining the structures in the heme environment of these globins. Picosecond time-resolved resonance Raman (ps-TR3) spectroscopy of transient five-coordinate heme species produced by the photolysis of carbon monoxide (CO) adducts of Cgb and Ngb showed Fe-His stretching (nu(Fe-His)) bands at 229 and 221 cm(-1), respectively. No time-dependent shift in the nu(Fe-His) band of Cgb and Ngb was detected in the 20-1000 ps time domain, in contrast to the case of myoglobin (Mb). These spectroscopic data, combined with previously reported crystallographic data, suggest that the structure of the heme pocket in Cgb and Ngb is altered upon CO binding in a manner different from that of Mb and that the scales of the structural alteration are different for Cgb and Ngb. The structural property of the heme distal side of the ligand-bound forms was investigated by observing the sets of (nu(Fe-CO), nu(C-O), delta(Fe-C-O)) and (nu(Fe-NO), nu(N-O), delta(Fe-N-O)) for the CO and nitric oxide (NO) complexes of Cgb and Ngb. A comparison of the spectra of some distal mutants of Cgb (H81A, H81V, R84A, R84K, and R84T) and Ngb (H64A, H64V, K67A, K67R, and K67T) showed that the CO adducts of Cgb and Ngb contained three conformers and that the distal His (His81 in Cgb and His64 in Ngb) mainly contributes to the interconversion of the conformers. These structural characteristics of Cgb and Ngb are discussed in relation to their ligand binding and physiological properties.

Comparison of the Dehalogenation of Dihalomethanes (CH2XI, where X = Cl, Br, I) Following Ultraviolet Photolysis in Aqueous and NaCl Saltwater Environments

The ultraviolet photolysis of low concentrations of CH(2)XI (X = Cl, Br, I) were investigated in water and saltwater solutions by photochemistry and picosecond time-resolved resonance Raman spectroscopy. Photolysis in both kinds of solutions formed mostly CH(2)(OH)(2) and HI and HX products. However, photolysis of the CH(2)XI molecules in saltwater resulted in production of some CH(2)XCl products not observed in aqueous solutions without salt present. The appearance of these new products in saltwater solutions is accompanied by a decrease in the amount of CH(2)(OH)(2), HI, and HX products compared to photolysis in aqueous solutions without salt present. The possible implications for photolysis of CH(2)XI and other polyhalomethanes in seawater and other salt aqueous environments compared to nonsaltwater solvated environments is briefly discussed.

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.

Water-Catalyzed O−H Insertion/HI Elimination Reactions of Isodihalomethanes (CH2X−I, Where X = Cl, Br, I) with Water and the Dehalogenation of Dihalomethanes in Water-Solvated Environments

A combined experimental and theoretical investigation of the ultraviolet photolysis of CH2XI (where X = Cl, Br, I) dihalomethanes in water is presented. Ultraviolet photolysis of low concentrations of CH2XI (where X = Cl, Br, I) in water appears to lead to almost complete conversion into CH2(OH)2 and HX and HI products. Picosecond time-resolved resonance Raman (ps-TR3) spectroscopy experiments revealed that noticeable amounts of CH2X-I isodihalomethane intermediates were formed within several picoseconds after photolysis of the CH2XI parent compound in mixed aqueous solutions. The ps-TR3 experiments in mixed aqueous solutions revealed that the decay of the CH2X-I isodihalomethane intermediates become significantly shorter as the water concentration increases, indicating that the CH2X-I intermediates may be reacting with water. Ab initio calculations found that the CH2X-I intermediates are able to react relatively easily with water via a water-catalyzed O-H insertion/HI elimination reaction to produce CH2X(OH) and HI products, with the barrier for these reactions increasing as X changes from Cl to Br to I. The ab initio calculations also found that the CH2X(OH) product can undergo a water-catalyzed HX elimination reaction to make H2C=O and HX products, with the barrier to reaction decreasing as X changes from Cl to Br to I. The preceding two water-catalyzed reactions produce the HI and HX leaving groups observed experimentally, and the H2C=O product further reacts with water to make the other CH2(OH)2 product observed in the photochemistry experiments. This suggests that that the CH2X-I intermediates react with water to form the CH2(OH)2 and HI and HX products observed in the photochemistry experiments. Ultraviolet photolysis of CH2XI (where X = Cl, Br, I) at low concentrations in water-solvated environments appears to lead to efficient dehalogenation and release of two strong acid leaving groups. We very briefly discuss the potential influence of this photochemistry in water on the decomposition of polyhalomethanes and halomethanols in aqueous environments.

Comparison of the dehalogenation of polyhalomethanes and production of strong acids in aqueous and salt (NaCl) water environments: Ultraviolet photolysis of CH2I2

The ultraviolet photolysis of CH(2)I(2) was studied in water and salt water solutions using photochemistry and picosecond time-resolved resonance Raman spectroscopy. Photolysis in both types of environments produces mainly CH(2)(OH)(2) and HI products. However, photolysis of CH(2)I(2) in salt water leads to the formation of different products/intermediates (CH(2)ICl and Cl(2) (-)) not observed in the absence of salt in aqueous solutions. The amount of CH(2)(OH)(2) and HI products appears to decrease after photolysis of CH(2)I(2) in salt water compared to pure water. We briefly discuss possible implications of these results for photolysis of CH(2)I(2) and other polyhalomethanes in sea water and other salt aqueous environments compared to nonsalt water solvated environments.

Time-Resolved Resonance Raman Study of the Triplet State of the p-Hydroxyphenacyl Acetate Model Phototrigger Compound

Picosecond and nanosecond time-resolved resonance Raman spectroscopy have been used to study the dynamics and structure of the phototrigger compound p-hydroxyphenacyl acetate (HPA) in acetonitrile solution. An intermediate was observed that exhibited significant quenching by oxygen. This intermediate was identified and attributed to the ππ* triplet state of HPA. Temporal evolution at early picosecond times indicates rapid intersystem crossing conversion and subsequent relaxation of the excess energy of the initially formed triplet state. B3LYP/6-311G** density functional theory (DFT) calculations were done to determine the structures and vibrational frequencies for both the HPA triplet and ground states. The experimental and theoretical results were compared and discussed in relation to the initial pathway for the p-hydroxyphenacyl deprotection reaction and the nature of the triplet state relative to other aromatic carbonyl compounds. The present results demonstrate the utility of applying ultrafast vibrational spectroscopy to study the mechanism of the photorelease process in phototrigger compounds.

Efficient dehalogenation of polyhalomethanes and production of strong acids in aqueous environments: water-catalyzed O--H-insertion and HI-elimination reactions of isodiiodomethane (CH2I-I) with water.

A combined experimental and theoretical study of the ultraviolet photolysis of CH2I2 in water is reported. Ultraviolet photolysis of low concentrations of CH2I2 in water was experimentally observed to lead to almost complete conversion into CH2(OH)2 and 2HI products. Picosecond time-resolved resonance Raman spectroscopy experiments in mixed water/acetonitrile solvents (25%-75% water) showed that appreciable amounts of isodiiodomethane (CH2I-I) were formed within several picoseconds and the decay of the CH2I-I species became substantially shorter with increasing water concentration, suggesting that CH2I-I may be reacting with water. Ab initio calculations demonstrate the CH2I-I species is able to react readily with water via a water-catalyzed O--H-insertion and HI-elimination reaction followed by its CH2I(OH) product undergoing a further water-catalyzed HI-elimination reaction to make a H2C=O product. These HI-elimination reactions produce the two HI leaving groups observed experimentally and the H2C=O product further reacts with water to produce the other final CH2(OH)2 product observed in the photochemistry experiments. These results suggest that CH2I-I is the species that reacts with water to produce the CH2(OH)2 and 2HI products seen in the photochemistry experiments. The present study demonstrates that ultraviolet photolysis of CH2I2 at low concentration leads to efficient dehalogenation and release of multiple strong acid (HI) leaving groups. Some possible ramifications for the decomposition of polyhalomethanes and halomethanols in aqueous environments as well as the photochemistry of polyhalomethanes in the natural environment are briefly discussed.

Ultrafast photoinduced electron transfer from N, N, N′, N′-tetramethyl- p-phenylenediamine and N, N, N′, N′-tetramethylbenzidine to dichloromethane

The photoinduced reaction of electron transfer (ET) from N, N, N′, N′-tetramethyl- p-phenylenediamine (TMPD) and N, N, N′, N′-tetramethylbenzidine (TMB) to the dichloromethane solvent has been studied by picosecond transient absorption and time-resolved resonance Raman spectroscopy. In neat CH 2Cl 2, “static” quenching of the lowest excited singlet state (S 1) of these amines is demonstrated. For TMPD, the reaction shows two kinetic contributions ( τ 1<4 ps, τ 2=9.5 ps) that are ascribed to the fractions of excited molecules that react before and after they are vibrationally relaxed, respectively. A faster quenching reaction is observed for TMB ( τ<4 ps) although it is less favourable energetically than for TMPD. This result is ascribed to a stronger acceptor–donor electronic coupling in the case of TMB, principally due a larger delocalization of the S 1 state donor orbital. The observation of a partial back electron transfer (9% yield) is tentatively explained as resulting from an evolution of the reaction product from an initial [TMB + , CH 2Cl 2 − ] ion pair allowing charge recombination to a final form [TMB + , Cl −] inappropriate to back ET.

Excited-state characters and dynamics of [W(CO)(5)(4-cyanopyridine)] and [W(CO)(5)(piperidine)] studied by picosecond time-resolved IR and resonance Raman spectroscopy and DFT calculations: roles of W --> L and W --> CO MLCT and LF excited states revised.

The characters, dynamics, and relaxation pathways of low-lying excited states of the complexes [W(CO)(5)L] [L = 4-cyanopyridine (pyCN) and piperidine (pip)] were investigated using theoretical and spectroscopic methods. DFT calculations revealed the delocalized character of chemically and spectroscopicaly relevant molecular orbitals and the presence of a low-lying manifold of CO pi-based unoccupied molecular orbitals. Traditional ligand-field arguments are not applicable. The lowest excited states of [W(CO)(5)(pyCN)] are W --> pyCN MLCT in character. They are closely followed in energy by W --> CO MLCT states. Excitation at 400 or 500 nm populates the (3)MLCT(pyCN) excited state, which was characterized by picosecond time-resolved IR and resonance Raman spectroscopy. Excited-state vibrations were assigned using DFT calculations. The (3)MLCT(pyCN) excited state is initially formed highly excited in low-frequency vibrations which cool with time constants between 1 and 20 ps, depending on the excitation wavelength, solvent, and particular high-frequency nu(CO) or nu(CN) mode. The lowest excited states of [W(CO)(5)(pip)] are W --> CO MLCT, as revealed by TD-DFT interpretation of a nanosecond time-resolved IR spectrum that was measured earlier in a low-temperature glass (Johnson, F. P. A.; George, M. W.; Morrison, S. L.; Turner, J. J. J. Chem. Soc., Chem. Commun. 1995, 391-393). MLCT(CO) excitation involves transfer of electron density from the W atom and, to a lesser extent, the trans CO to the pi orbitals of the four cis CO ligands. Optical excitation into MLCT(CO) transition of either complex in fluid solution triggers femtosecond dissociation of a W-N bond, producing [W(CO)(5)(solvent)]. It is initially vibrationally excited both in nu(CO) and anharmonicaly coupled low-frequency modes. Vibrational cooling occurs with time constants of 16-22 ps while the intramolecular vibrational energy redistribution from the v = 1 nu(CO) modes is much slower, 160-220 ps. No LF excited states have been found for the complexes studied in a spectroscopically relevant range up to 6-7 eV. It follows that spectroscopy, photophysics, and photochemistry of [W(CO)(5)L] and related complexes are well described by an interplay of close-lying MLCT(L) and MLCT(CO) excited states. The high-lying LF states play only an indirect photochemical role by modifying potential energy curves of MLCT(CO) states, making them dissociative.

Direct observation of an isopolyhalomethane O-H insertion reaction with water: picosecond time-resolved resonance Raman (ps-TR3) study of the isobromoform reaction with water to produce a CHBr2OH product.

Picosecond time-resolved resonance Raman (ps-TR3) spectroscopy was used to obtain the first definitive spectroscopic observation of an isopolyhalomethane O-H insertion reaction with water. The ps-TR3 spectra show that isobromoform is produced within several picoseconds after photolysis of CHBr3 and then reacts on the hundreds of picosecond time scale with water to produce a CHBr2OH reaction product. Photolysis of low concentrations of bromoform in aqueous solution resulted in noticeable formation of HBr strong acid. Ab initio calculations show that isobromoform can react with water to produce a CHBr2(OH) O-H insertion reaction product and a HBr leaving group. This is consistent with both the ps-TR3 experiments that observe the reaction of isobromoform with water to form a CHBr2(OH) product and photolysis experiments that show HBr acid formation. We briefly discuss the implications of these results for the phase dependent behavior of polyhalomethane photochemistry in the gas phase versus water solvated environments.

Tracing "loose electrons" by picosecond time-resolved resonance Raman spectroscopy with near-infrared excitation

Tracing "loose electrons" by picosecond time-resolved resonance Raman spectroscopy with near-infrared excitation

Solvent-dependent dynamics of the MQ*-->ReII excited-state electron transfer in [Re(MQ+)(CO)3(dmb)]2+.

The Re-->MQ(+) MLCT excited state of [Re(MQ(+))(CO)(3)(dmb)](2+) (MQ(+) = N-methyl-4,4'-bipyridinium, dmb = 4,4'-dimethyl-2,2'-bipyridine), which is populated upon 400-nm irradiation, was characterized by picosecond time-resolved IR and resonance Raman spectroscopy, which indicate large structural differences relative to the ground state. The Re-->MQ(+) MLCT excited state can be formulated as [Re(II)(MQ*)(CO)(3)(dmb)](2+). It decays to the ground state by a MQ*-->Re(II) back-electron transfer, whose time constant is moderately dependent on the molecular nature of the solvent, instead of its bulk parameters: formamides approximately DMSO approximately MeOH (1.2-2.2 ns) < THF, aliphatic nitriles (3.2-3.9 ns) << ethylene-glycol approximately 2-ethoxyethanol (4.2-4.8 ns) < pyridine (5.7 ns) < MeOCH(2)CH(2)OMe (6.9 ns) < PhCN (7.5 ns) < MeNO(2) (8.6 ns) <<< CH(2)Cl(2), ClCH(2)CH(2)Cl (25.9-28.9 ns). An approximate correlation was found between the back-reaction rate constant and the Gutmann donor number. Temperature dependence of the decay rate measured in CH(2)Cl(2), MeOH, and BuCN indicates that the inverted MQ*-->Re(II) back-electron transfer populates a manifold of higher vibrational levels of the ground state. The solvent dependence of the electron transfer rate is explained by solvent effects on inner reorganization energy and on frequencies of electron-accepting vibrations, by interactions between the positively charged MQ(+) pyridinium ring and solvent molecules in the electron-transfer product, that is the [Re(MQ(+))(CO)(3)(dmb)](2+) ground state.

Picosecond dynamics of G-protein coupled receptor activation in rhodopsin from time-resolved UV resonance Raman spectroscopy.

The protein response to retinal chromophore isomerization in the visual pigment rhodopsin is studied using picosecond time-resolved UV resonance Raman spectroscopy. High signal-to-noise Raman spectra are obtained using a 1 kHz Ti:Sapphire laser apparatus that provides <3 ps visible (466 nm) pump and UV (233 nm) probe pulses. When there is no time delay between the pump and probe events, tryptophan modes W18, W16, and W3 exhibit decreased Raman scattering intensity. At longer pump-probe time delays of +5 and +20 ps, both tryptophan (W18, W16, W3, and W1) and tyrosine (Y1 + 2xY16a, Y7a, Y8a) peak intensities drop by up to 3%. These intensity changes are attributed to decreased hydrophobicity in the microenvironment near at least one tryptophan and one tyrosine residue that likely arise from weakened interaction with the beta-ionone ring of the chromophore following cis-to-trans isomerization. Examination of the crystal structure suggests that W265 and Y268 are responsible for these signals. These UV Raman spectral changes are nearly identical to those observed for the rhodopsin-to-Meta I transition, implying that impulsively driven protein motion by the isomerizing chromophore during the 200 fs primary transition drives key structural changes that lead to protein activation.