Primary Photochemical Event Research Articles

Computational Studies of the Primary Phototransduction Event in Visual Rhodopsin

This Account addresses recent advances in the elucidation of the detailed molecular rearrangements due to the primary photochemical event in rhodopsin, a prototypical G-protein-coupled receptor (GPCR) responsible for the signal transmission cascade in the vertebrate vision process. The reviewed studies provide fundamental insight on long-standing problems regarding the assembly and function of the individual residues and bound water molecules that form the rhodopsin active site, a center that catalyzes the 11-cis/all-trans isomerization of the retinyl chromophore in the primary step of the phototransduction mechanism. Emphasis is placed on the authors' recent computational studies, based on state-of-the-art quantum mechanics/molecular mechanics (QM/MM) hybrid methods, addressing the structural refinement of the retinyl chromophore binding site in high-resolution X-ray structures of bovine visual rhodopsin, the energy storage mechanism, and the molecular origin of spectroscopic changes due to the primary photochemical event.

Hydrogen abstraction from solvents by the triplet state of p-benzoquinone: a time-resolved electron paramagnetic resonance and laser flash photolysis study

The reaction of triplet p-benzoquinone in several solvents, such as ethanol, 2-propanol, ethylene glycol, t-butanol, 1,4-dioxane, tetrahydrofuran and cyclohexane, has been studied. The primary photochemical event was shown to be a hydrogen atom abstraction from the solvent and not an electron transfer. In the time-resolved EPR experiments, in all cases, except t-butanol and cyclohexane, the radicals from the solvent were recorded along with p-benzosemiquinone radical and the spectra were dominated by triplet mechanism of spin polarization, giving totally emissive EPR signals. The sites of abstracted hydrogen atom have been identified. In particular, whereas the methine hydrogen atom is abstracted from 2-propanol, both the methylene and hydroxyl hydrogen atoms are abstracted from ethanol. From laser flash photolysis experiments, the rate constants of the hydrogen abstraction in all solvents, except t-butanol and cyclohexane, were found to be (1–5) × 108 M−1 s−1.

QM/MM Study of Energy Storage and Molecular Rearrangements Due to the Primary Event in Vision

The energy storage and the molecular rearrangements due to the primary photochemical event in rhodopsin are investigated by using quantum mechanics/molecular mechanics hybrid methods in conjunction with high-resolution structural data of bovine visual rhodopsin. The analysis of the reactant and product molecular structures reveals the energy storage mechanism as determined by the detailed molecular rearrangements of the retinyl chromophore, including rotation of the ϕ(C11–C12) dihedral angle from −11° in the 11- cis isomer to −161° in the all- trans product, where the preferential sense of rotation is determined by the steric interactions between Ala-117 and the polyene chain at the C13 position, torsion of the polyene chain due to steric constraints in the binding pocket, and stretching of the salt bridge between the protonated Schiff base and the Glu-113 counterion by reorientation of the polarized bonds that localize the net positive charge at the Schiff-base linkage. The energy storage, computed at the ONIOM electronic-embedding approach (B3LYP/6-31G*:AMBER) level of theory and the S 0 → S 1 electronic-excitation energies for the dark and product states, obtained at the ONIOM electronic-embedding approach (TD-B3LYP/6-31G*//B3LYP/6-31G*:AMBER) level of theory, are in very good agreement with experimental data. These results are particularly relevant to the development of a first-principles understanding of the structure-function relations in prototypical G-protein-coupled receptors.

Subpicosecond spectroscopic studies on the photochemical events of 2-dimethylaminoethanethiol-capped CdS nanoparticles in water

In order to confirm further the charge effect of capping agent on the photochemical events of the surface-capped CdS particles, the colloidal solution of 2-dimethylaminoethanethiol (ET)-capped CdS particles (ET-CdS) was prepared and its subpicosecond time-resolved transient absorption was measured. ET has a positive charge in its protonated form. From the comparison with the data for TG-CdS (TG: 1-thioglycerol), measured under same experimental conditions, the influence of the positive charge of the capping agent on the behavior and decay dynamics of the photo-generated electron and hole was investigated. It was suggested from these results that ET which was used as a capping agent in the present study was protonated and then acted as an electron capturer rather than as a hole scavenger in neutral pH range. As a consequence, disappearance of the photo-generated electrons in the primary photochemical events of ET-CdS colloidal solutions was accelerated, compared with that for TG-CdS colloidal solutions. The result also suggests that the charge of capping agents on the particles significantly affects the behavior of electrons and holes generated by the photo-excitation of the particles. However, pH dependence was not observed for the transient absorption of ET-CdS and its decay dynamics. As the reason, it is considered that the proton-elimination equilibrium of the protonated ET capping on the surface of CdS cores are hardly affected by the change of pH of the solutions in the adjustable pH range, hence, the total electron trapping ability of capping agents on the particles also did not change significantly.

Influences of proton-dissociation equilibrium of capping agents on the photo-chemical events of the colloidal solutions containing the thiol-capped cadmium sulfide particles

The primary photo-chemical events of the aqueous colloidal solutions containing surface-modified CdS particles were investigated by two different methods, of which one employed subpicosecond spectroscopy to measure the transient absorption due to photo-generated electrons in the primary photo-chemical events, and the other stationary irradiation to measure their photo-stability. The pH dependence of transient absorption due to photo-generated electrons (trapped and hydrated electrons) in the colloidal solutions of thioglycerol (TG)-capped CdS (TG-CdS) and mercaptoacetate (MA)-capped CdS (MA-CdS) is similar to that of their photo-stabilities under the stationary irradiation, respectively. However, there was large difference in the pH dependence between TG-CdS and MA-CdS. Namely, the absorption intensity and the photo-stability decrease with lowering pH of the solutions for MA-CdS in the pH range lower than 7, where the negative charge of a carboxyl group of MA is neutralized by addition of proton. However, absorption intensity and photo-stability were hardly influenced by pH of the solution in whole pH range in the case of TG-CdS. These results suggest that the negative charge of the capping agents acts advantageously on the hole-trapping process in the primary photo-chemical events of the surface-modified CdS particles and their photo-stability.

Direct Measurement of the Photoelectric Response Time of Bacteriorhodopsin via Electro-Optic Sampling

The photovoltaic signal associated with the primary photochemical event in an oriented bacteriorhodopsin film is measured by directly probing the electric field in the bacteriorhodopsin film using an ultrafast electro-optic sampling technique. The inherent response time is limited only by the laser pulse width of 500 fs, and permits a measurement of the photovoltage with a bandwidth of better than 350 GHz. All previous published studies have been carried out with bandwidths of 50 GHz or lower. We observe a charge buildup with an exponential formation time of 1.68 ± 0.05 ps and an initial decay time of 31.7 ps. Deconvolution with a 500-fs Gaussian excitation pulse reduces the exponential formation time to 1.61 ± 0.04 ps. The photovoltaic signal continues to rise for 4.5 ps after excitation, and the voltage profile corresponds well with the population dynamics of the K state. The origin of the fast photovoltage is assigned to the partial isomerization of the chromophore and the coupled motion of the Arg-82 residue during the primary event.

A quantitative structure–function relationship for the Photosystem II reaction center: Supermolecular behavior in natural photosynthesis

Light-induced charge separation is the primary photochemical event of photosynthesis. Efficient charge separation in photosynthetic reaction centers requires the balancing of electron and excitation energy transfer processes, and in Photosystem II (PSII), these processes are particularly closely entangled. Calculations that treat the cofactors of the PSII reaction center as a supermolecular complex allow energy and electron transfer reactions to be described in a unified way. This calculational approach is shown to be in good agreement with experimentally observed energy and electron transfer dynamics. This supermolecular view also correctly predicts the effect of changing the redox potentials of cofactors by site-directed mutagenesis, thus providing a unified and quantitative structure-function relationship for the PSII reaction center.

Nature of the Surface Crossing Process in Bacteriorhodopsin: Computer Simulations of the Quantum Dynamics of the Primary Photochemical Event

The quantum dynamics of the primary photoisomerization event in bacteriorhodopsin is studied by a semiclassical trajectory approach. The relevant surface crossing probability is evaluated from the wave functions and potential surfaces of a hybrid quantum mechanical/molecular mechanics (QM/MM) Hamiltonian of the complete chromophore−protein−solvent system. The QM/MM model combines consistently the quantum mechanical Hamiltonian of the chromophore with the microscopic electric field of the ionized groups and induced dipoles of the protein−solvent system. The QCFF/PI Hamiltonian of the chromophore is adjusted to reproduce relevant ab initio results. The nonadiabatic coupling term is calculated numerically from the corresponding wave functions. The simulations are performed by combining the ENZYMIX and QCFF/PI molecular modeling programs. The effect of the protein on the absorption spectrum of the chromophore is examined. It is found that this spectrum reflects the effect of the protein permanent ...

Primary photoreaction of photoactive yellow protein studied by subpicosecond-nanosecond spectroscopy.

The primary photochemical event of photoactive yellow protein (PYP) was studied by laser flash photolysis experiments on a subpicosecond-nanosecond time scale. PYP was excited by a 390-nm pulse, and the transient difference absorption spectra were recorded by a multichannel spectrometer for a more reliable spectral analysis than previously possible. Just after excitation, an absorbance decrease due to the stimulated emission at 500 nm and photoconversion of PYP at 450 nm were observed. The stimulated emission gradually shifted to 520 nm and was retained up to 4 ps. Then, the formation of a red-shifted intermediate with a broad absorption spectrum was observed from 20 ps to 1 ns. Another red-shifted intermediate with a narrow absorption spectrum was formed after 2 ns and was stable for at least 5 ns. The latter is therefore believed to correspond to I1 (PYP(L)), which has been detected on a nanosecond time scale or trapped at -80 degrees C. Singular value decomposition analysis demonstrated that the spectral shifts observed from 0.5 ps to 5 ns could be explained by two-component decay of excited state(s) and conversion from PYP(B) to PYP(L). The amount of PYP(L) at 5 ns was less than that of photoconverted PYP, suggesting the formation of another intermediate, PYP(H). In addition, the absorption spectra of these intermediates were calculated based on the proposed reaction scheme. Together, these results indicate that the photocycle of PYP at room temperature has a branched pathway in the early stage and is essentially similar to that observed under low-temperature spectroscopy.

Thermoluminescence in Chloroplasts as an Indicator of Alterations in Photosystem 2 Reaction Centre by Biotic and Abiotic Stresses

Thermoluminescence (TL) in green plants arises from charge recombination of charged molecules in the reaction centre (RC) of photosystem 2 (PS2) in chloroplasts. The TL technique is used for detection of alterations in the architecture of PS2 RCs. The donor side 'S-states' and the acceptor side quinone molecules (QA and QB) are involved the charge recombination processes of PS2. High temperature (70–75 °C) glow peaks are also used to detect non-photosynthetic peroxidation processes in thylakoid membranes. The TL peaks with their characteristic charge recombination can be utilised for the study of chloroplast development, ageing, chemical, biotic, and abiotic stress induced alterations in the PS2 RC and for the study of the primary photochemical events of photosynthesis. The technique has been used successfully in the characterisation of transgenic plants in the study of genetically engineered organisms.

A nuclear-encoded protein of prokaryotic origin is essential for the stability of photosystem II in Arabidopsis thaliana.

To understand the regulatory mechanisms underlying the biogenesis of photosystem II (PSII) we have characterized the nuclear mutant hcf136 of Arabidopsis thaliana and isolated the affected gene. The mutant is devoid of any photosystem II activity, and none of the nuclear- and plastome-encoded subunits of this photosystem accumulate to significant levels. Protein labelling studies in the presence of cycloheximide showed that the plastome-encoded PSII subunits are synthesized but are not stable. The HCF136 gene was isolated by virtue of its T-DNA tag, and its identity was confirmed by complementation of homozygous hcf136 seedlings. Immunoblot analysis of fractionated chloroplasts showed that the HCF136 protein is a lumenal protein, found only in stromal thylakoid lamellae. The HCF136 protein is produced already in dark-grown seedlings and its levels do not increase dramatically during light-induced greening. This accumulation profile confirms the mutational data by showing that the HCF136 protein must be present when PSII complexes are made. HCF136 homologues are found in the cyanobacterium Synechocystis species PCC6803 (slr2034) and the cyanelle genome of Cyanophora paradoxa (ORF333), but are lacking in the plastomes of chlorophytes and metaphytes as well as from those of rhodo- and chromophytes. We conclude that HCF136 encodes a stability and/or assembly factor of PSII which dates back to the cyanobacterial-like endosymbiont that led to the plastids of the present photosynthetic eukaryotes.

Subpicosecond studies of primary photochemical events of CdS particles with surface modified by various capping agents

Q-CdS particles with surface modified by thiophenol (PhS) or 2-mercaptobenzoxazole (MBO) were prepared and their time-resolved transient absorption spectra were measured in acetonitrile in subpicosecond to picosecond time domain in order to investigate the influences of capping agents on primary photochemical events such as electron and hole trapping processes and decay kinetics of the trapped charge carriers on capped semiconductor Q-particles. In addition, time-resolved transient absorption spectra of aqueous CdS colloidal solutions containing sodium hexametaphosphate (HMP) as a stabilizer against flocculation were also measured in the same time domain and compared to obtain knowledge about the trap sites for electrons photo-produced on capped CdS particles in acetonitrile. The shape and the decay profile of transient absorption spectra obtained by subpicosecond laser flash photolysis of PhS-CdS and MBO-CdS in acetonitrile are markedly different from those of non-capped CdS particles in acetonitrile obtained previously using picosecond laser flash photolysis by Karnat et al. The results suggest that capping agents such as PhS and MBO influence significantly hole trapping process and decay dynamics of the trapped charge carriers on the capped CdS. Furthermore, it seems that the trap site of the electrons generated by laser excitation of PhS-CdS or MBO-CdS in acetonitrile is mainly at the interface of particle-solution and the possibility for the production of solvated electrons is low because the shapes and decay kinetics of the transient absorption spectra of PhS-CdS or MBO-CdS in acetonitrile are clearly different from those HMP-CdS in water in which the photo-production of the hydrated electrons is known.

Laser pulse energy requirements for remote sensing of chlorophyll fluorescence

When measuring laser induced chlorophyll fluorescence the intention is to probe the photosynthetic system of the plant, that is, to measure its state without notably affecting it. This is a common measuring principle. It was not known if the 10 ns green laser pulses used for our laser induced fluorescence measurements satisfy this requirement. In this article we consider the problem from a theoretical point of view and by experiment. Two unwanted effects may play a role: unintended partial closure of the reaction centers and exciton annihilations. The primary photochemical events in the photosystem of the plant are simulated numerically. In addition the fuorescence in the dark adapted state has been measured with a range of excitation energies. Both theoretical and experimental results are compared and analyzed. Our results show that the probing laser pulse, used for measuring the laser induced chlorophyll fluorescence, should not exceed an excitation energy of 100 mJ/m 2 at the plant. Additional laser pulses that could sere as an actinic light source to close all reaction centers before the measurement must have a pulse length of at least 100 ns.

Ab initio molecular dynamics of rhodopsin

Abstract

A resonance Raman study of the C=N configurations of octopus rhodopsin, bathorhodopsin, and isorhodopsin.

The resonance Raman spectra of octopus rhodopsin, bathorhodopsin, and isorhodopsin at 120 K have been obtained as well as those of pigments regenerated with isotopically labeled retinals near the C14-C15 bond. Deuteration of the Schiff base nitrogen induces relatively large changes in the C-C stretch region between 1100 and 1300 cm-1, including a large frequency shift of the C14-C15 stretch mode located at 1206-1227 cm-1 in the three octopus species, as revealed by the Raman spectra of their 14,15-(13)C2 derivatives. Such results are different compared to those of the bovine pigments, in which no significant frequency shift of the C14-C15 stretch mode was observed upon Schiff base N deuteration. In an earlier Raman study of a Schiff base model compound which contained only one single bond adjacent to two double bonds, we have found that the stretch mode of this C-C single bond at 1232 cm-1 shifts up by 15 cm-1 and its intensity is also greatly reduced upon Schiff base N deuteration when the C=N configuration is anti [Deng et al., (1994) J. Phys. Chem. 98, 4776-4779]. The same study has also shown that when the C=N configuration is syn, the C-C stretch mode should be at about 1150 cm-1. Since the C14-C15 stretch mode frequency is relatively high in the spectra of octopus rhodopsin and bathorhodopsin (> 1200 cm-1) and since the normal mode pattern near the Schiff base is similar to the model, we suggest that the C=N configuration in these two species is anti. The different responses of the C14-C15 stretch mode to the Schiff base nitrogen deuteration in bovine and octopus pigments are due to the fact that the coupled C14-C15 stretch and the C12-C13 stretch motions in the model compound or in bovine rhodopsin are altered in octopus rhodopsin so that the stretch motion of the C14-15 bond is more localized, similar to the C-C stretch motion in the small Schiff base model compound. In clear contrast with the bovine rhodopsin Raman spectrum, which is very similar to that for the 11-cis-retinal Schiff base, the drastically different octopus rhodopsin spectrum indicates large protein perturbations on the C11=C12-C13 moiety, either by steric or by electrostatic interactions. Further studies are required to determine if such spectral differences indicate a difference of the energy conversion mechanism in the primary photochemical event of these two pigments.

Photochromic poly(α-amino acid)s: photomodulation of molecular and supramolecular structure

Photochromic polymers are able to respond to light giving reversible variations of their structure and conformation which, in turn, are accompanied by variations of their physical and chemical properties. This paper describes the photochromic behaviour of poly(α-amino acid)s containing azobenzene and spiropyran groups in the side chains, and illustrates the most significative examples of their photoresponse effects. Experimental findings provide evidence that the molecular and supramolecular structure of these polypeptides can be photomodulated using the photosensitive side chains as effectors. The extent and the kind of photoresponse, including photoinduced coil ⇌ α-helix transitions, photostimulated aggregation/disaggregation processes, reversible variations of viscosity and solubility, were found to depend on polymer and environment conditions. Interpretative models of the photoresponse effects indicate that photochromic poly(α-amino acid) systems actually behave as amplifiers and transducers of the primary photochemical event occurring in the side chains, and therefore may be promising materials for application in optical technology.

Ultrafast Spectroscopy of Rhodopsins — Photochemistry at Its Best!

AbstractRecent advances in laser technology now allow us to study the ultrafast primary photochemical events in rhodopsin, bacteriorhodopsin, and halorhodopsin in real time. The results of various ultrafast studies of rhodopsins are reviewed with an emphasis on (1) the relationship between the reaction rate and reaction efficiency and its implications for the mechanism of isomerization, (2) the homogeneity of the reaction pathways, and (3) the role of the protein in the reaction dynamics. The results mandate the introduction of a new paradigm to describe these ultrafast reactions that specifically considers the importance of the timescale of vibrational dephasing and relaxation relative to the reactive motion and the contribution of vibrational coherence to the reaction mechanism.

Steric constraints in the retinal binding pocket of sensory rhodopsin I

Steric constraints in the retinal binding pocket of sensory rhodopsin I (SR-I) are analyzed by studying effects of sample temperature and retinal analogs. The flash-induced yield of the earliest detected intermediate S610, which corresponds to the K intermediate in the bacteriorhodopsin (BR) photocycle, decreases below 220 K and reaches zero at 100 K, while K formation is independent of temperature. The reduced S610 formation at low temperatures indicates a more restricted retinal binding pocket in SR-I during primary photochemical events. Introduction of bulky substituents on the retinal polyene chain in four retinal analogs greatly retards or blocks the final step of chromophore binding to the apoprotein of SR-I. Except for the 14-methyl substitution, these modifications exhibit little or no effect on chromophore binding to BR apoprotein. These results corroborate that the retinal polyene chain binding domain in SR-I is more sterically constrained than that of the retinal pocket in BR. Deletion of the beta-ionone ring renders the analog SR-I pigments nonfunctional, as does deletion of the 13-methyl group, but the corresponding BR analogs are both photochemically and physiologically active. In contrast to the corresponding BR analog, photolysis of the analog SR-I reconstituted with 13-desmethylretinal does not produce an S610-like intermediate at room temperature. The above results and the previous findings that protein constraints inhibit the accommodation of a stable 13-cis-retinal configuration in SR-I suggest a model in which the 13-methyl group functions as a fulcrum to permit movement of one or both ends of retinal to overcome an energy barrier against isomerization.

Comparative study of primary photochemical events of two retinal proteins, bacteriorhodopsin and halorhodopsin, by use of subpicosecond time-resolved spectroscopy

The primary photochemical events of bacteriorhodopsin (bR) were compared with those of halorhodopsin (hR) by subpicosecond time-resolved spectroscopy. The absorption and stimulated emission spectra display similar features. It is proposed that the isomerization reaction and relaxation to the fluorescent state occur simultaneously from the Franck—Condon excited state. The quantum yield of cis—trans isomerization in bR is about twice that in hR.

Photoisomerization mechanism of the rhodopsin chromophore: picosecond photolysis of pigment containing 11-cis-locked eight-membered ring retinal.

The primary photochemical event in rhodopsin is an 11-cis to 11-trans photoisomerization of its retinylidene chromophore to form the primary intermediate photorhodopsin. Earlier picosecond studies have shown that no intermediate is formed when the retinal 11-ene is fixed through a bridging five-membered ring, whereas a photorhodopsin-like intermediate is formed when it is fixed through a flexible seven-membered ring. Results from a rhodopsin analog formed from a retinal with locked 11-ene structure through the more flexible eight-membered ring (Ret8) are described. Incubation of bovine opsin with Ret8 formed two pigments absorbing at 425 nm (P425) and 500 nm (P500). P425, however, is an artifact because it formed from thermally denatured opsin or other proteins and Ret8. Excitation of P500 with a picosecond green pulse led to formation of two intermediates corresponding to photo- and bathorhodopsins. These results demonstrate that an appearance of early intermediates is dependent on the flexibility of the 11-ene and that the photoisomerization of P500 proceeds by stepwise changes of chromophore-protein interaction, which in turn leads to a relaxation of the highly twisted all-trans-retinylidene chromophore in photorhodopsin.