TIME-RESOLVED RESONANCE RAMAN STUDIES ON THE PHOTOCHEMICAL CYCLE OF BACTERIORHODOPSIN

Abstract— Kinetic resonance Raman (RR) experiments were designed to study the time-behaviour of the retinal-binding protein bacteriorhodopsin (BR) in its photochemical cycle. The unphotolyzed chro-mophore B-570 and the two intermediates L -550 and M-412 were probed by the characteristic C=C stretching vibrations of the retinal moiety. Time resolution was achieved with a spinning cell as flow system in combination with two CW lasers in a pump-probe configuration. RR spectra were probed at 475 nm at various delay times between pump and probe event. The deconvolution of the spectra into the various components B-570, L-550 and M-412 was carried out by curve fitting procedures. It was found that at pH7.4 L-550 decays — with a time-constant of 62 μs — not completely but to a residual level of 35% of its initial value. This intermediate L -amplitude finally disappears in the ms-range (4.5 ms) synchroneously with the intermediate M -412. An analogeous time-behaviour was found at pH 4.6. In the basic range also an " L " -intermediate could be identified which is coupled to the long-lived M-component. To explain the peculiar time-dependence it is proposed that during the fast decay of L a dynamic equilibrium between L and M is established. Then during the reconstitution of B -570 the two intermediates disappear synchroneously. A molecular model is presented in which the dynamic equilibrium between L and M is explained by an oscillatory motion of a proton from the Schiff base group of the chromophore to its counterion.     

ULTRAVIOLET RESONANCE RAMAN SPECTROSCOPY OF BACTERIORHODOPSIN

Ultraviolet resonance Raman spectra of bacteriorhodopsin have been obtained using 229 nm excitation from a hydrogen-shifted neodymium yttrium aluminum garnet (Nd: YAG) laser. High signal-to-noise spectra are observed exhibiting vibrational bands at 762, 877, 1011, 1175, 1356, 1552 and 1617 cm-1 which are assigned to scattering from tryptophan and tyrosine side chains. This demonstrates the feasibility of using UV resonance Raman spectroscopy to monitor aromatic amino acid structural changes during the bacteriorhodopsin photocycle.     

POWER- AND TIME-RESOLVED RESONANCE RAMAN STUDIES AND CONFORMATIONAL CHANGES IN BACTERIORHODOPSIN

Abstract— By comparing the resonance Raman spectra of the retinal of the intermediates of bacteriorho-dopsin (obtained by using fixed flow with residence of time of 10 ps. variable laser power and frequency as well as computer subtraction techniques) with those of model compounds and with each other, the following possible conclusions can be obtained: (1) There exists stronger interaction between the retinal and the opsin in bacteriorhodopsin than that present in rhodopsin. (2) Conformational changes seem to take place during the dark light adaptation process as well as during the photosynthetic cycle. (3) The appearance of the spectrum of the retinal in the fingerprint region for the bL₅₅₀ and bM₄₁₂ intermediates is similar despite large shifts in their optical absorption maxima. This might argue against the theory that proposes ground state retinal conformational changes to explain the observed red shift in the optical spectra of retinal upon combining with the opsin. (4) Contrary to bM ₄₁₂ , the bL₅₅₀ species seems to be protonated. The fact that loss of proton does not seem to change the retinal conformation greatly might suggest that the protein and its ionic environment might carry the larger share of the load in the deprotonation process.     

LOW TEMPERATURE RESONANCE RAMAN STUDY OF THE L INTERMEDIATE OF BACTERIORHODOPSIN

Abstract— Resonance Raman spectra of photostationary state mixtures at — 80°C of bR568, L, L', and M for light-adapted bacteriorhodopsin were obtained. The approximate Raman spectrum of L + L' was obtained by computer, subtracting the known Raman spectra of bR568 and M from the photostationary state spectra. Several new species not previously observed with the bacteriorhodopsin photocycle have been proposed recently. We compare our results to these studies in order to investigate the photoreaction scheme of bacteriorhodopsin.     

TIME-RESOLVED RESONANCE RAMAN SPECTRA OF THE PHOTOCYCLE INTERMEDIATES OF ACID AND DEIONIZED BACTERIORHODOPSIN

Abstract— The photocycle of bacteriorhodopsin (bR) and its perturbed forms are investigated by a time-resolved resonance Raman study. These experiments were performed in the C=C stretching and in the fingerprint spectral regions for the acid blue, acid purple and deionized forms of bR.
The main observations are as follows: (1) isomerization of the retinal, from all- trans to 13- cis , occurs in native bR and in all of the acid and deionized perturbed bR species; (2) formation of the early intermediates (the K₆₁₀ and L₅₅₀ analogues) also occur in native bR and in all of the perturbed species; and (3) deprotonation of the protonated Schiff base (PSB), to give the M₄₁₂ type intermediate, occurs in native bR, but is inhibited in all of the perturbed bR species on the time-scale of the native bR photocycle.
The results show that isomerization alone is not a prerequisite for the PSB deprotonation process. The observed photocycle, initiated with retinal isomerization, is found to occur from all- trans to 13- cis in all of the perturbed forms of bR. In addition, the results imply that removal of the cations, of an increase in the hydrogen ion concentration, prevent only the PSB deprotonation process and not the formation of earlier cycle intermediates. Some attention is focused on the two blue forms of bR (acid and deionized) due to the fact that their ground-state absorption maximum, unphotolyzed Raman spectra, and Raman spectra changes during the photocycle are all very similar. The similarities between the acid blue and deionized blue forms in the fingerprint region support previous suggestions that both blue species have nearly the same retinal active site.     

RESONANCE RAMAN AND SURFACE-ENHANCED RESONANCE RAMAN SPECTROSCOPY OF HYPERICIN

Hypericin has been found to exhibit a variety of photodynamic effects. To correlate biological activity with molecular structure, complete physical characterization of hypericin is required. The vibrational spectrum has been determined and resonance Raman and surface enhanced resonance Raman scattering spectra are reported. In addition, the Raman spectrum of a model compound has been studied to facilitate assignment of the vibrational modes of hypericin.     

PRIMARY PHOTOCHEMISTRY OF BACTERIORHODOPSIN: COMPARISON OF FOURIER TRANSFORM INFRARED DIFFERENCE SPECTRA WITH RESONANCE RAMAN SPECTRA

Abstract —Fourier transform infrared (FTIR) difference spectra of the BR→rK transition in bacteriorhodopsin at 77→K are compared with analogous resonance Raman difference spectra obtained using a spinning sample cell at 77→K. The vibrational frequencies observed in the FTIR spectra of native purple membrane and of purple membrane regenerated with 15-deuterioretinal are in good agreement with the frequencies observed in the Raman spectra, indicating that the lines in the FTIR difference spectra arise predominantly from retinal chromophore vibrations. This agreement confirms that the spinning cell method for obtaining resonance Raman spectra of K minimizes potential contributions from unwanted photoproducts. The unexpected similarity between the resonance Raman scattering intensities and the FTIR absorption intensities for BR and K is discussed in terms of the delocalized electronic structure of the chromophore. Finally, comparison of the Schiff base regions of the K Raman and FTIR spectra provide additional information on the assignment of its Schiff base vibration.     

TIME-RESOLVED RESONANCE RAMAN SPECTROSCOPY OF THE BACTERIORHODOPSIN PHOTOCYCLE ON THE PICOSECOND AND NANOSECOND TIME SCALES

Abstract— Resonance Raman spectra of the picosecond bacteriorhodopsin intermediate(s) have been obtained by microbeam, flow and subtraction techniques using a synchronously pumped, cavity-dumped dye laser. Nanosecond spectra also were measured with this laser by cavity dumping without mode-locking. The picosecond spectra in the fingerprint region, which is sensitive to the configuration of the retinal chromophore, differ from spectra of the parent bR₅₇₀ but could be correlated to the spectrum of bR^DA₅₅₀ , a “13-cis” species which has been determined from spectra of bR₅₇₀ and bR^DA₅₆₀. The picosecond transient and bR^DA₅₅₀ also are similar in the 950–1050 cm^-1“deuteration fingerprint” region when the medium is changed from H₂O to D₂O. These results suggest that trans—cis isomerization occurs during the 40-ps pulse duration. The shift relative to the parent bR₅₇₀ in the ethylenic stretch region suggests that the picosecond and nanosecond transients absorb at wavelengths longer than 570 nm. The C band at 1646 cm^-1 is found to shift or to broaden upon photolysis in the picosecond time scale. This might suggest a change in the electronic structure of the group and its environment on the picosecond time domain. The nanosecond spectra obtained in this work (with 15-ns pulses) are similar to the spectra previously observed on the 100-ns time scale but are slightly different from the picosecond spectrum. These data suggest that more than one transient species appears on the picosecond-to-nanosecond time scale. The temporal evolution of Raman bands in the fingerprint as well as the low energy (950–1050 cm^-1) region and its implications are discussed.     

RESONANCE RAMAN SPECTRA OF BACTERIORHODOPSIN MUTANTS WITH SUBSTITUTIONS AT ASP-85, ASP-96, AND ARG-82

Detergent solubilized bacteriorhodopsin (BR) proteins which contain alterations made by site-directed mutagenesis (Asp-96----Asn, D96N; Asp-85----Asn, D85N; and Arg-82----Gln, R82Q) have been studied with resonance Raman spectroscopy. Raman spectra of the light-adapted (BRLA) and M species in D96N are identical to those of native BR, indicating that this residue is not located near the chromophore. The BRLA states of D85N and especially R82Q contain more of the 13-cis, C = N syn (BR555) species under ambient illumination compared to solubilized native BR. Replacement of Asp-85 with Asn causes a 25 nm red-shift of the absorption maximum and a frequency decrease in both the ethylenic (-7 cm-1) and the Schiff base C = NH+ (-3 cm-1) stretching modes of BRLA. These changes indicate that Asp-85 is located close to the protonated retinal Schiff base. The BRLA spectrum of R82Q exhibits a slight perturbation of the C = NH+ band, but its M spectrum is unperturbed. The Raman spectra and the absorption properties of D85N and R82Q suggest that the protein counterion environment involves the residues Asp-85-, Arg-82+ and presumably Asp-212-. These data are consistent with a model where the strength of the protein-chromophore interaction and hence the absorption maximum depends on the overall charge of the Schiff base counterion environment.     

稀土催化聚乙炔的拉曼光谱研究

比较了用稀土和白川催化体系,制备的70％顺式含量及全反式聚乙炔的拉曼光谱及其随激发光源波长的变化。报道了用稀土催化体系制备的可溶性聚乙炔的拉曼光谱。这些实验结果支持导致反式聚乙炔拉吏异常的主要原因是反式聚乙炔链段共轭长度分散的解释。     

A DETAILED RESONANCE RAMAN STUDY OF THE M412 INTERMEDIATE IN THE BACTERIORHODOPSIN PHOTOCYCLE

Abstract— Resonance Raman spectra of various M₄₁₂ species associated with the bacteriorhodopsin photocycle have been obtained. These correspond to the two forms observed during the formation of M₄1₂ and the two forms that are observed during its decay in absorption experimeents. We do not see any significant difference between the Raman spectra of any of these forms. We therefore conclude that the differences in these species are due to the differences in the protein structure and not in the chromophore.     

对苯二胺和N-取代衍生物正离子自由基的共振喇曼光谱研究

本文中报道了对苯二胺和四种N-烷基取代衍生物正离子自由基的共振喇曼光谱, 揭示了自由基是具有明显C=C和C=N双键性质的半醌式结构, N原子上给电子基团的取代引起上述化学键振动峰低频移动, 反映出结构一端N上有烷基的自由基中与取代基直接相连的N原子失去一个p电子带正电荷, 并吸引环上的π电子形成一定程度C=N双键. 共振喇曼光谱随激发光波长的变化表明, 自由基的两个可见电子吸收带分别主要产生于环结构的π→π*和包含N原子结构的n→π*电子跃迁。     

呫吨类染料的表面增强共振喇曼散射光谱

本文研究了呫吨类染料分子(R110, RB, SRB, R_6G, R101和SF)吸附于化学沉积银岛膜上的表面增强共振喇曼散射(SERRS)光谱。文中详细的介绍了获得SERRS的实验条件, 对谱线的归属作了指认, 并讨论了共振、表面增强、吸附状态和衬底制备条件等因素对SERRS的特征和对喇曼散射截面的增强的影响。结果表明, SERRS是研究强荧光染料分子以及它们与金属表面相互作用的一种新的有效手段。     

RAMAN STUDIES OF AEROSOL CHEMICAL REACTIONS

Since Professor Matijevité and his colleagues published pioneering work on aerosol chemical reactions, based on experiments with monodisperse aerosol generators and laminar flow reactors, there has been considerable progress in the chemical characterization of aerosol particles and the study of their chemical reactions. This paper surveys recent developments and new research on the application of Raman spectroscopy to gas/liquid and gas/solid aerosol reactions. Of particular interest are applications of the vibrating orifice aerosol generator and electrodynamic and optical levitators coupled to Raman spectrometers to explore aerosol chemistry. The systems examined include the production of polymeric microsphcrcs, the generation of metal oxide particles from alkoxide droplets, SQ2/sorbent particle reactions used for demilitarization of stick gases, chemical characterization of particle arrays, and reactions following collisions of dissimilar particles. The complications associated with the interpretation of Raman data introduced by morphology-dependent resonances in the elastically scattered light are also examined.     

吩噻嗪和吩噁嗪氧化中间体的共振喇曼光谱研究

本文对吩噻嗪和吩噁嗪的单电子氧化产生的两种氧化态的亚稳态正离子进行了共振喇曼光谱研究。随着吩噻嗪的p电子逐步失去,结构变形振动δ_(CNC)和δ_(CSC)的喇曼频移显著地增大,环C=C伸缩振动区域的喇曼峰数目明显变多。由此推断,随着吩噻嗪正离子氧化态的增高,正离子转变成平面共轭结构。     

RESONANCE RAMAN SPECTRA AND MOLECULAR ORIENTATION DISTRIBUTION OF INTERFACIAL MONOLAYERS

An orientation distribution model has been suggested for interfacial monomolecular lay-ers by taking account of the orientational uniaxiality of long-chain amphiphilic molecules inmonolayers. The resonance Raman spectra of azo-containing monolayers are the oreticallytreated on the basis of the model. Order parameters and (P_4> are obtained from polar-ized Raman measurements, giving rise to distribution graphs. These analyses indicate thatthe orientation distribution alters upon the increase of adsorption amount and displays inter-esting variation among different interfacial monolayers. The aggregate aspects of the 2-dimensional condensed states are also discussed from the viewpoint of molecular orientation.     

RESONANCE RAMAN SPECTRA OF THE Pr-FORM OF PHYTOCHROME

Abstract— Resonance Raman spectra of the P_r-form of oat phytochrome have been obtained at 77 K. Interference from phytochrome fluorescence is avoided by employing far-red 752 nm excitation. Vibrational assignments are suggested for the tetrapyrrole chromophore in phytochrome by comparison with previously published model compound spectra and by examining the characteristic shifts induced by deuteration of the pyrrole nitrogens. The lack of carbonyl intensity, the frequencies of the 1626 and 1644 cm^-1 C=C stretching modes, and the presence of an intense mode at 1326 cm^-1 are all consistent with a protonated structure for the tetrapyrrole chromophore in P_r. This suggests that the -50 nm red-shift of the protein-bound chromophore absorption compared to the chromophore in vitro is caused by protonation of the pyrrole nitrogen.