Ground-state Intermediates Research Articles

Unveiling Solvation Dynamics of Excited and Ground States via Ultrafast Pump-Dump-Probe Spectroscopy.

The conventional ultrafast pump-probe spectroscopy has primarily focused on examining the formation and decay of the excited state intermediates, but it is very difficult to detect those intermediates while the formation is slow and dissipation is much fast because of the limited concentration during the intrinsic photocycle. To address this issue, a multipulse ultrafast pump-dump-probe spectroscopy was employed to generate and probe the short-lived ground state intermediates (GSIs) in an electronic push-pull pyrene derivative (EPP). This particular derivative undergoes planarized intramolecular charge transfer (PICT) in the excited state upon initial femtosecond pulse excitation. After applying the dump pulse once the PICT was formed, the blue-shifted transient absorption GSIs with the ground state dynamics of the structure recovery was directly observed. It is found that GSIs undergo slower reorganization than the PICT formation in the excited state of EPP due to the solvation effect with different dipole moments of ground states and excited states. These findings provide a comprehensive understanding of the full photocycle dynamics of both the ground and excited states, shedding light on the presence of hidden ground state behaviors.

Is orange carotenoid protein photoactivation a single-photon process?

In all published photoactivation mechanisms of orange carotenoid protein (OCP), absorption of a single photon by the orange dark state starts a cascade of red-shifted OCP ground-state intermediates that subsequently decay within hundreds of milliseconds, resulting in the formation of the final red form OCPR, which is the biologically active form that plays a key role in cyanobacteria photoprotection. A major challenge in deducing the photoactivation mechanism is to create a uniform description explaining both single-pulse excitation experiments, involving single-photon absorption, and continuous light irradiation experiments, where the red-shifted OCP intermediate species may undergo re-excitation. We thus investigated photoactivation of Synechocystis OCP using stationary irradiation light with a biologically relevant photon flux density coupled with nanosecond laser pulse excitation. The kinetics of photoactivation upon continuous and nanosecond pulse irradiation light show that the OCPR formation quantum yield increases with photon flux density; thus, a simple single-photon model cannot describe the data recorded for OCP in vitro. The results strongly suggest a consecutive absorption of two photons involving a red intermediate with ≈100 millisecond lifetime. This intermediate is required in the photoactivation mechanism and formation of the red active form OCPR.

In Silico Discovery of Multistep Chemistry Initiated by a Conical Intersection: The Challenging Case of Donor-Acceptor Stenhouse Adducts.

Detailed mechanistic understanding of multistep chemical reactions triggered by internal conversion via a conical intersection is a challenging task that emphasizes limitations in theoretical and experimental techniques. We present a discovery-based, hypothesis-free computational approach based on first-principles molecular dynamics to discover and refine the switching mechanism of donor-acceptor Stenhouse adducts (DASAs). We simulate the photochemical experiment in silico, following the "hot" ground state dynamics for 10 ps after photoexcitation. Using state-of-the-art graphical processing units-enabled electronic structure calculations we performed in total ∼2 ns of nonadiabatic ab initio molecular dynamics discovering (a) critical intermediates that are involved in the open-to-closed transformation, (b) several competing pathways which lower the overall switching yield, and (c) key elements for future design strategies. Our dynamics describe the natural evolution of both the nuclear and electronic degrees of freedom that govern the interconversion between DASA ground-state intermediates, exposing significant elements for future design strategies of molecular switches.

Photoinduced Chromophore Hydration in the Fluorescent Protein Dreiklang Is Triggered by Ultrafast Excited-State Proton Transfer Coupled to a Low-Frequency Vibration.

Because of growing applications in advanced fluorescence imaging, the mechanisms and dynamics of photoinduced reactions in reversibly photoswitchable fluorescent proteins are currently attracting much interest. We report the first time-resolved study of the photoswitching of Dreiklang, so far the only fluorescent protein to undergo reversible photoinduced chromophore hydration. Using broadband femtosecond transient absorption spectroscopy, we show that the reaction is triggered by an ultrafast deprotonation of the chromophore phenol group in the excited state in 100 fs. This primary step is accompanied by coherent oscillations that we assign to its coupling with a low-frequency mode, possibly a deformation of the chromophore hydrogen bond network. A ground-state intermediate is formed in the picosecond-nanosecond regime that we tentatively assign to the deprotonated water adduct. We suggest that proton ejection from the phenol group leads to a charge transfer from the phenol to the imidazolinone ring, which triggers imidazolinone protonation by nearby Glu222 and catalyzes the addition of the water molecule.

Ultrafast Carbonyl Motion of the Photoactive Yellow Protein Chromophore Probed by Femtosecond Circular Dichroism

Motions of the trans-p-coumaric acid carbonyl group following the photoexcitation of the R52Q mutant of photoactive yellow protein (PYP) are investigated, for the first time, by ultrafast time-resolved circular dichroism (TRCD) spectroscopy. TRCD is monitored in the near-ultraviolet, over a time scale of 10 ps. Immediately after excitation, TRCD is found to exhibit a large negative peak, which decays within a few picoseconds. A quantitative analysis of the signals shows that, upon excitation, the carbonyl group undergoes a fast (≪0.8 ps) and unidirectional flipping motion in the excited state with an angle of ca. 17-53°. For the subset of proteins that do not enter the signaling photocycle, TRCD provides strong evidence that the carbonyl group moves back to its initial position, leading to the formation of a nonreactive ground-state intermediate of trans conformation. The initial ground state is then restored within ca. 3 ps. Comparative study of R52Q and wild-type PYP provides direct evidence that the absence of Arg52 has no effect on the conformational changes of the chromophore during those steps.

Reactive Ground-State Pathways Are Not Ubiquitous in Red/Green Cyanobacteriochromes

Recent characterization of the red/green cyanobacteriochrome (CBCR) NpR6012g4 revealed a high quantum yield for its forward photoreaction [J. Am. Chem. Soc. 2012, 134, 130-133] that was ascribed to the activity of hidden, productive ground-state intermediates. The dynamics of the pathways involving these ground-state intermediates was resolved with femtosecond dispersed pump-dump-probe spectroscopy, the first such study reported for any CBCR. To address the ubiquity of such second-chance initiation dynamics (SCID) in CBCRs, we examined the closely related red/green CBCR NpF2164g6 from Nostoc punctiforme. Both NpF2164g6 and NpR6012g4 use phycocyanobilin as the chromophore precursor and exhibit similar excited-state dynamics. However, NpF2164g6 exhibits a lower quantum yield of 32% for the generation of the isomerized Lumi-R primary photoproduct, compared to 40% for NpR6012g4. This difference arises from significantly different ground-state dynamics between the two proteins, with the SCID mechanism deactivated in NpF2164g6. We present an integrated inhomogeneous target model that self-consistently fits the pump-probe and pump-dump-probe signals for both forward and reverse photoreactions in both proteins. This work demonstrates that reactive ground-state intermediates are not ubiquitous phenomena in CBCRs.

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.

ChemInform Abstract: Synthesis of Proline-Valine Pseudodipeptide Enol Lactones, Serine Protease Inhibitors.

Pseudodipeptides of proline-valine that incorporate protio or halo enol lactone moieties have been synthesized from common acetylenic acid precursors; in each case, two diasteromers were prepared in enantiomerically pure form. The preparation began with isomeric propargylic alcohols derived from L-prolinal, which are further elaborated into the methyleneoxy valine pseudodipeptide analogues via an oxalactam intermediate. The pseudodipeptide acetylenic acids could by cyclized to the protio enol lactones by mercuric salts and could be elaborated to tetrapeptide analogues either before or after cyclization. The relative stability of the two diastereomeric enol lactone systems toward intramolecular acyl transfer could be rationalized by molecular mechanics energy calculations on ground-state and tetrahedral intermediates believed to be involved in the reaction. While the halo enol lactones derived from the pseudotetrapeptides proved to be very unstable, they could be prepared from the n-butyl carbamate derivatives of the dipeptide

Mid-Infrared Picosecond Pump−Dump−Probe and Pump−Repump-Probe Experiments to Resolve a Ground-State Intermediate in Cyanobacterial Phytochrome Cph1

Multipulse picosecond mid-infrared spectroscopy has been used to study photochemical reactions of the cyanobacterial phytochrome photoreceptor Cph1. Different photophysical schemes have been discussed in the literature to describe the pathways after photoexcitation, particularly, to identify reaction phases that are linked to photoisomerisation and electronic decay in the 1566-1772 cm(-1) region that probes C=C and C=O stretching modes of the tetrapyrrole chromophore. Here, multipulse spectroscopy is employed, where, compared to conventional visible pump-mid-infrared probe spectroscopy, an additional visible pulse is incorporated that interacts with populations that are evolving on the excited- and ground-state potential energy surfaces. The time delays between the pump and the dump pulse are chosen such that the dump pulse interacts with different phases in the reaction process. The pump and dump pulses are at the same wavelength, 640 nm, and are resonant with the Pr ground state as well as with the excited state and intermediates. Because the dump pulse additionally pumps the remaining, partially recovered, and partially oriented ground-state population, theory is developed for estimating the fraction of excited-state molecules. The calculations take into account the model-dependent ground-state recovery fraction, the angular dependence of the population transfer resulting from the finite bleach that occurs with linearly polarized intense femtosecond optical excitation, and the partially oriented population for the dump field. Distinct differences between the results from the experiments that use a 1 or a 14 ps dump time favor a branching evolution from S1 to an excited state or reconfigured chromophore and to a newly identified ground-state intermediate (GSI). Optical dumping at 1 ps shows the instantaneous induced absorption of a delocalized C=C stretching mode at 1608 cm(-1), where the increased cross section is associated with the electronic ground-state structure of the ZZZ configuration of the linear tetrapyrrole chromophore. The dump-induced absorption decays with time constants of 5 and 19 ps to the Pr ground state. Employing a dump pulse at 14 ps results in an instantaneous decrease of the absorption of the 1608 cm(-1) band, indicating repumping of the GSI. The dump-induced absorption recovers back to the GSI with a 6 ps lifetime. A spectral similarity is observed between the 6 ps phase in the dump experiment and the 3 ps component found in the two-pulse pump-probe measurement. Combined with the dominance of ground-state absorption bands in the dump-induced spectrum, this indicates the presence of a GSI, which is additionally characterized by previously unidentified induced absorption at 1710 and 1570-80 cm(-1). The metastable photoproduct Lumi-R, which is in the electronic ground state and populated at 500 ps after excitation of Pr, is highly efficiently repumped into the Pr ground state with the power density used. After repumping, Lumi-R is not recovered on the 500 ps time scale of the experiment and is distinct from the GSI of Pr since it is not associated with its characteristic induced absorption at 1710 and 1570-80 cm(-1).

Femtosecond Kinetics of Photoconversion of the Higher Plant Photoreceptor Phytochrome Carrying Native and Modified Chromophores

The photoprocesses of native (phyA of oat), and of C-terminally truncated recombinant phytochromes, assembled instead of the native phytochromobilin with phycocyanobilin (PCB-65 kDa-phy) and iso-phycocyanobilin (iso-PCB-65 kDa-phy) chromophores, have been studied by femtosecond transient absorption spectroscopy in both their red absorbing phytochrome (P r) and far-red absorbing phytochrome (P fr) forms. Native P r phytochrome shows an excitation wavelength dependence of the kinetics with three main picosecond components. The formation kinetics of the first ground-state intermediate I 700, absorbing at ∼690 nm, is mainly described by 28 ps or 40 ps components in native and PCB phytochrome, respectively, whereas additional ∼15 and 50 ps components describe conformational dynamics and equilibria among different local minima on the excited-state hypersurface. No significant amount of I 700 formation can be observed on our timescale for iso-PCB phytochrome. We suggest that iso-PCB-65 kDa-phy either interacts with the protein differently leading to a more twisted and/or less protonated configuration, or undergoes P r to P fr isomerization primarily via a different configurational pathway, largely circumventing I 700 as an intermediate. The isomerization process is accompanied by strong coherent oscillations due to wavepacket motion on the excited-state surface for both phytochrome forms. The femto- to (sub-)nanosecond kinetics of the P fr forms is again quite similar for the native and the PCB phytochromes. After an ultrafast excited-state relaxation within ∼150 fs, the chromophores return to the first ground-state intermediate in 400–800 fs followed by two additional ground-state intermediates which are formed with 2–3 ps and ∼400 ps lifetimes. We call the first ground-state intermediate in native phytochrome I fr·750, due to its pronounced absorption at that wavelength. The other intermediates are termed I fr·675 and pseudo-P r. The absorption spectrum of the latter already closely resembles the absorption of the P r chromophore. PCB-65 kDa-phy shows a very similar kinetics, although many of the detailed spectral features in the transients seen in native phy are blurred, presumably due to wider inhomogeneous distribution of the chromophore conformation. Iso-PCB-65 kDa-phy shows similar features to the PCB-65 kDa-phy, with some additional blue-shift of the transient spectra of ∼10 nm. The sub-200 fs component is, however, absent, and the picosecond lifetimes are somewhat longer than in 124 kDa phytochrome or in PCB-65 kDa-phy. We interpret the data within the framework of two- and three-dimensional potential energy surface diagrams for the photoisomerization processes and the ground-state intermediates involved in the two photoconversions.

Ultrafast infrared spectroscopy reveals a key step for successful entry into the photocycle for photoactive yellow protein

Photoactive proteins such as PYP (photoactive yellow protein) are generally accepted as model systems for studying protein signal state formation. PYP is a blue-light sensor from the bacterium Halorhodospira halophila. The formation of PYP's signaling state is initiated by trans-cis isomerization of the p-coumaric acid chromophore upon the absorption of light. The quantum yield of signaling state formation is approximately 0.3. Using femtosecond visible pump/mid-IR probe spectroscopy, we investigated the structure of the very short-lived ground state intermediate (GSI) that results from an unsuccessful attempt to enter the photocycle. This intermediate and the first stable GSI on pathway into the photocycle, I0, both have a mid-IR difference spectrum that is characteristic of a cis isomer, but only the I0 intermediate has a chromophore with a broken hydrogen bond with the backbone N atom of Cys-69. We suggest, therefore, that breaking this hydrogen bond is decisive for a successful entry into the photocycle. The chromophore also engages in a hydrogen-bonding network by means of its phenolate group with residues Tyr-42 and Glu-46. We have investigated the role of this hydrogen bond by exchanging the H bond-donating residue Glu-46 with the weaker H bond-donating glutamine (i.e., Gln-46). We have observed that this mutant exhibits virtually identical kinetics and product yields as WT PYP, even though during the I0-to-I1 transition, on the 800-ps time scale, the hydrogen bond of the chromophore with Gln-46 is broken, whereas this hydrogen bond remains intact with Glu-46.

Photochemistry of 1,2,4-Oxadiazoles. A DFT Study on Photoinduced Competitive Rearrangements of 3-Amino- and 3-N-Methylamino-5-perfluoroalkyl-1,2,4-oxadiazoles

The photoinduced competitive rearrangements of 5-perfluoroalkyl-3-amino(N-alkylamino)-1,2,4-oxadiazoles have been investigated by DFT calculations and UV-vis spectroscopy. The observed product selectivity depends on the number of hydrogen atoms present in the amino moiety and involves two or three possible routes: (i) ring contraction-ring expansion (RCRE), (ii) internal-cyclization isomerization (ICI), or (iii) C3-N2 migration-nucleophilic attack-cyclization (MNAC). UV absorption and fluorescence spectra of the reactants, and vertical excitation energy values, calculated by time dependent DFT, support the involvement of a neutral singlet excited state in the photoexcitation process. The values of the standard free energy of the most stable prototropic tautomers of reactant, products, proposed reaction intermediates, and deprotonated anionic transition states allowed us to rationalize the competition among the three rearrangements, in agreement with chemical trapping experiments, in terms of: (i) the evolution of the excited state toward three stable ground-state intermediates, (ii) tautomeric and deprotonation equilibria occurring in methanol solution for each intermediate, and (iii) relative stabilization of intermediates and transition states in the thermally driven section of the reaction.

Uncovering the hidden ground state of green fluorescent protein

The fluorescence properties of GFP are strongly influenced by the protonation states of its chromophore and nearby amino acid side chains. In the ground state, the GFP chromophore is neutral and absorbs in the near UV. Upon excitation, the chromophore is deprotonated, and the resulting anionic chromophore emits its green fluorescence. So far, only excited-state intermediates have been observed in the GFP photocycle. We have used ultrafast multipulse control spectroscopy to prepare and directly observe GFP's hidden anionic ground-state intermediates as an integral part of the photocycle. Combined with dispersed multichannel detection and advanced global analysis techniques, the existence of two distinct anionic ground-state intermediates, I(1) and I(2), has been unveiled. I(1) and I(2) absorb at 500 and 497 nm, respectively, and interconvert on a picosecond timescale. The I(2) intermediate has a lifetime of 400 ps, corresponding to a proton back-transfer process that regenerates the neutral ground state. Hydrogen/deuterium exchange of the protein leads to a significant increase of the I(1) and I(2) lifetimes, indicating that proton motion underlies their dynamics. We thus have assessed the complete chain of reaction intermediates and associated timescales that constitute the photocycle of GFP. Many elementary processes in biology rely on proton transfers that are limited by slow diffusional events, which seriously precludes their characterization. We have resolved the true reaction rate of a proton transfer in the molecular ground state of GFP, and our results may thus aid in the development of a generic understanding of proton transfer in biology.

Photoisomerization and Photoionization of the Photoactive Yellow Protein Chromophore in Solution

Dispersed pump-dump-probe spectroscopy has the ability to characterize and identify the underlying ultrafast dynamical processes in complicated chemical and biological systems. This technique builds on traditional pump-probe techniques by exploring both ground- and excited-state dynamics and characterizing the connectivity between constituent transient states. We have used the dispersed pump-dump-probe technique to investigate the ground-state dynamics and competing excited-state processes in the excitation-induced ultrafast dynamics of thiomethyl p-coumaric acid, a model chromophore for the photoreceptor photoactive yellow protein. Our results demonstrate the parallel formation of two relaxation pathways (with multiple transient states) that jointly lead to two different types of photochemistry: cis- trans isomerization and detachment of a hydrated electron. The relative transition rates and quantum yields of both pathways have been determined. We find that the relaxation of the photoexcited chromophores involves multiple, transient ground-state intermediates and the chromophore in solution does not generate persistent photoisomerized products, but instead undergoes photoionization resulting in the generation of detached electrons and radicals. These results are of great value in interpreting the more complex dynamical changes in the optical properties of the photoactive yellow protein.

A photodissociation study of HgI2

The photodissociation of jet-cooled HgI2 has been studied in the 244–323 nm region using a tunable ultraviolet (UV) laser. Dispersed emission, action, and multiphoton ionization spectra have been recorded. A vibrational analysis of the action spectra near 305 nm shows that the production of excited Hg atoms from HgI2 UV photodissociation proceeds through a HgI ground-state intermediate. Near 248 nm the results are less conclusive. Possible reasons for a wavelength-dependent kinetic mechanism are discussed.

Photochemical transformations. 28. Comparisons of "ionic" intermediates produced photochemically with corresponding ground-state intermediates. Further studies in some chlorobenzobicyclooctadienyl systems

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTPhotochemical transformations. 28. Comparisons of "ionic" intermediates produced photochemically with corresponding ground-state intermediates. Further studies in some chlorobenzobicyclooctadienyl systemsStanley J. Cristol and Robert M. StromCite this: J. Am. Chem. Soc. 1980, 102, 17, 5577–5580Publication Date (Print):August 1, 1980Publication History Published online1 May 2002Published inissue 1 August 1980https://doi.org/10.1021/ja00537a027RIGHTS & PERMISSIONSArticle Views32Altmetric-Citations4LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (529 KB) Get e-Alerts Get e-Alerts

Photochemical transformations. 24. Comparison of ionic intermediates produced photochemically with corresponding ground-state intermediates. Initial studies in some chlorobenzooctadienyl systems

Photochemical transformations. 24. Comparison of ionic intermediates produced photochemically with corresponding ground-state intermediates. Initial studies in some chlorobenzooctadienyl systems

Photosensitised reactions of α-pinene

The photosensitised decomposition of α-pinene in the liquid phase, using acetophenone or propiophenone as the sensitiser, yields cis- and trans-β-ocimenes as the only products. This is in contrast to pyrolysis, radiolysis, and direct photolysis, where mixtures of products are obtained. Benzophenone does not act as a photosensitiser. It is possible that the reaction involves a vibrationally excited ground-state intermediate. The initial polychromatic quantum yield, (λ= 280–366 mµ), with acetophenone as sensitiser is 0·8.