Excitonic energy level structure and pigment-protein interactions in the recombinant water-soluble chlorophyll protein. II. Spectral hole-burning experiments.

Persistent spectral hole burning at 4.5 K has been used to investigate the excitonic energy level structure and the excited state dynamics of the recombinant class-IIa water-soluble chlorophyll-binding protein (WSCP) from cauliflower. The hole-burned spectra are composed of four main features: (i) a narrow zero-phonon hole (ZPH) at the burn wavelength, (ii) a number of vibrational ZPHs, (iii) a broad low-energy hole at ~665 and ~683 nm for chlorophyll b- and chlorophyll a-WSCP, respectively, and (iv) a second satellite hole at ~658 and ~673 nm for chlorophyll b- and chlorophyll a-WSCP, respectively. The doublet of broad satellite holes is assigned to an excitonically coupled chlorophyll dimer. The lower-energy holes at ~665 and ~683 nm for chlorophyll b- and chlorophyll a-WSCP, respectively, represent the lower exciton states. Taking into account the parameters of electron-phonon coupling, the lower exciton state can be assigned as the fluorescence origin. The lower exciton state is populated by two processes: (i) exciton relaxation from the higher exciton state and (ii) vibrational relaxation within the lower exciton state. Assuming identical site energies for the two excitonically coupled chlorophyll molecules, the dipole-dipole interaction energy J is directly determined to be 85 and 100 cm(-1) for chlorophyll b- and chlorophyll a-WSCP, respectively, based on the positions of the satellite holes. The Gaussian low-energy absorption band identified by constant fluence hole burning at 4.5 K has a width of ~150 cm(-1) and peaks at 664.9 and 682.7 nm for chlorophyll b- and chlorophyll a-WSCP, respectively. The action spectrum is broader and blue-shifted compared to the fluorescent lower exciton state. This finding can be explained by a slow protein relaxation between energetically inequivalent conformational substates within the lowest exciton state in agreement with the results of Schmitt et al. (J. Phys. Chem. B2008, 112, 13951).

Water soluble chlorophyll binding protein of higher plants: a most suitable model system for basic analyses of pigment-pigment and pigment-protein interactions in chlorophyll protein complexes.

This short review paper describes spectroscopic studies on pigment-pigment and pigment-protein interactions of chlorophyll (Chl) a and b bound to the recombinant protein of class IIa water soluble chlorophyll protein (WSCP) from cauliflower. Two Chls form a strongly excitonically coupled open sandwich dimer within the tetrameric protein matrix. In marked contrast to the mode of excitonic coupling of Chl and bacterio-Chl molecules in light harvesting complexes and reaction centers of all photosynthetic organisms, the unique structural pigment array in the Chl dimer of WSCP gives rise to an upper excitonic state with a large oscillator strength. This property opens the way for thorough investigations on exciton relaxation processes in Chl-protein complexes. Lifetime measurements of excited singlet states show that the unusual stability towards photodamage of Chls bound to WSCP, which lack any protective carotenoid molecule, originates from a high diffusion barrier to interaction of molecular dioxygen with Chl triplets. Site selective spectroscopic methods provide a wealth of information on the interactions of the Chls with the protein matrix and on the vibronic structure of the pigments. The presented data and discussions illustrate the great potential of WSCP as a model system for systematic experimental and theoretical studies on the functionalizing of Chls by the protein matrix. It opens the way for further detailed analyses and a deeper understanding of the properties of pigment protein complexes.

Diode-pumped passively mode-locked Nd:GSAG laser at 942 nm.

Stable mode-locking of a diode-pumped Nd:GSAG laser emitting at 942 nm between the 4F2/3-4I9/2 transition has been demonstrated. With a z cavity and a semiconductor saturable absorber mirror passive mode locker, we obtained 8.7 ps pulses at repetition rate of 95.6 MHz and average output power of 510 mW. The total optical efficiency is about 3.1%.

Thermally activated superradiance and intersystem crossing in the water-soluble chlorophyll binding protein.

The crystal structure of the class IIb water-soluble chlorophyll binding protein (WSCP) from Lepidium virginicum is used to model linear absorption and circular dichroism spectra as well as excited state decay times of class IIa WSCP from cauliflower reconstituted with chlorophyll (Chl) a and Chl b. The close agreement between theory and experiment suggests that both types of WSCP share a common Chl binding motif, where the opening angle between pigment planes in class IIa WSCP should not differ by more than 10 degrees from that in class IIb. The experimentally observed (Schmitt et al. J. Phys. Chem. B 2008, 112, 13951) decrease in excited state lifetime of Chl a homodimers with increasing temperature is fully explained by thermally activated superradiance via the upper exciton state of the dimer. Whereas a temperature-independent intersystem crossing (ISC) rate is inferred for WSCP containing Chl a homodimers, that of WSCP with Chl b homodimers is found to increase above 100 K. Our quantum chemical/electrostatic calculations suggest that a thermally activated ISC via an excited triplet state T4 is responsible for the latter temperature dependence.

Excited state dynamics in recombinant water-soluble chlorophyll proteins (WSCP) from cauliflower investigated by transient fluorescence spectroscopy.

The present study describes the fluorescence emission properties of recombinant water-soluble chlorophyll (Chl) protein (WSCP) complexes reconstituted with either Chl a or Chl b alone (Chl a only or Chl b only WSCP, respectively) or mixtures of both pigments at different stoichiometrical ratios. Detailed investigations were performed with time and space correlated ps fluorescence spectroscopy within the temperature range from 10 to 295 K. The following points were found: (a) The emission spectra at room temperature (295 K) are well characterized by bands with a dominating Lorentzian profile broadened due to phonon scattering and peak positions located at 677, 684 and 693 nm in the case of Chl a only WSCP and at 665, 675 and 689 nm for Chl b only WSCP. In addition, all spectra contain minor bands in the longer wavelength region. (b) The emission spectra at 10 K of samples suspended in buffer containing 50% glycerol are dominated by bands peaking at 668 nm for Chl b only WSCP and at 685 nm for Chl a only WSCP and samples reconstituted with mixtures of Chl a and Chl b. (c) At 10 K and in buffer with 50% glycerol the decay kinetics of WSCP samples with Chl a only are dominated by a component with a time constant of 6.2 (+/-0.2) ns at 685 nm while those of WSCP containing mixtures of Chl a and Chl b are characterized by a slightly shorter value of 6.0 (+/-0.2) ns. WSCP containing Chl b only exhibits a distinctly longer value of 7.0 (+/-0.3) ns at an emission wavelength of 668 nm. (d) The decay associated emission spectra at 10 K of all samples exhibit at least 3 decay components with time constants of 80-120 ps, 2-4 ns and 6-7 ns in 50% glycerol. These results are consistently described within the framework of our previously presented model (J. Phys. Chem. B 2007, 111, No. 46, 13325; J. Phys. Chem. B 2007, 111, No. 35, 10487) , for the structural motifs of chlorophyll binding to the tetrameric protein matrix of WSCP. It is shown that formation of strongly coupled open sandwich dimers does not lead to quenching of 1Chl a* or 1Chl b*.

Pigment-pigment and pigment-protein interactions in recombinant water-soluble chlorophyll proteins (WSCP) from cauliflower.

Plants contain water-soluble chlorophyll-binding proteins (WSCPs) that function neither as antennas nor as components of light-induced electron transfer of photosynthesis but are likely constituents of regulatory protective pathways in particular under stress conditions. This study presents results on the spectroscopic properties of recombinant WSCP from cauliflower reconstituted with chlorophyll b (Chl b) alone or with mixtures of Chl a and Chl b. Two types of experiments were performed: (a) measurements of stationary absorption spectra at 77 and 298 K and CD spectra at 298 K and (b) monitoring of laser flash-induced transient absorption changes with a resolution of 200 fs in the time domain of up to 100 ps. On the basis of a theoretical analysis outlined by Renger et al. (J. Phys. Chem. B 2007, 111, 10487) the data obtained in part (a) are interpreted within a model where tetrameric WSCP binds predominantly two Chl molecules in the form of an excitonically coupled "open sandwich" dimer with a tilt angle of about 30 degrees between the chlorin planes. The time-resolved measurements on Chl a/Chl b heterodimers are described by two exponential kinetics with time constants of 400 fs and 7 ps. These kinetics are assumed to reflect a heterogeneous population of WSCPs with Chl dimers either in excitonic coupled "open sandwich" or weakly coupled geometric arrays. The 400 fs component is assigned to excited-state relaxations from the upper to the lower excitonic level of the strongly coupled "open sandwich" dimer, while the 7-8 ps component probably indicates excitation energy transfer from 1Chl b* to Chl a in a dimer array with weak coupling due to significantly longer mutual distances between the chlorin rings.

Refinement of a structural model of a pigment-protein complex by accurate optical line shape theory and experiments.

Time-local and time-nonlocal theories are used in combination with optical spectroscopy to characterize the water-soluble chlorophyll binding protein complex (WSCP) from cauliflower. The recombinant cauliflower WSCP complexes reconstituted with either chlorophyll b (Chl b) or Chl a/Chl b mixtures are characterized by absorption spectroscopy at 77 and 298 K and circular dichroism at 298 K. On the basis of the analysis of these spectra and spectra reported for recombinant WSCP reconstituted with Chl a only (Hughes, J. L.; Razeghifard, R.; Logue, M.; Oakley, A.; Wydrzynski, T.; Krausz, E. J. Am. Chem. Soc. U.S.A. 2006, 128, 3649), the "open-sandwich" model proposed for the structure of the pigment dimer is refined. Our calculations show that, for a reasonable description of the data, a reduction of the angle between pigment planes from 60 degrees of the original model to about 30 degrees is required when exciton relaxation-induced lifetime broadening is included in the analysis of optical spectra. The temperature dependence of the absorption spectrum is found to provide a unique test for the two non-Markovian theories of optical spectra. Based on our data and the 1.7 K spectra of Hughes et al. (2006), the time-local partial ordering prescription theory is shown to describe the experimental results over the whole temperature range between 1.7 K and room temperature, whereas the alternative time-nonlocal chronological ordering prescription theory fails at high temperatures. Modified-Redfield theory predicts sub-100 fs exciton relaxation times for the homodimers and a 450 fs time constant in the heterodimers. Whereas the simpler Redfield theory gives a similar time constant for the homodimers, the one for the heterodimers deviates strongly in the two theories. The difference is explained by multivibrational quanta transitions in the protein which are neglected in Redfield theory.

Raman crystal lasers in the visible and near-infrared.

Raman lasers based on potassium gadolinium tungstate and lead tungstate crystals pumped by a to approximately 120 ps Nd: YAG laser at 1.064 microm were developed. High reflection mirrors for the Stokes wavelength have been used to generate near-infrared and eye safe spectral region of 1.15 - 1.32 microm. Second harmonic generation of the generated Raman lasers was observed. Eifficient multiple Stokes and anti-Stokes picosecond generation in 64 crystals have been shown to exhibit stimulated Raman scattering on about 700 lines covering the whole visible and near-infrared spectrum. All stimulated Raman scattering (SRS) wavelengths in the visible and near-infrared spectrum are identified and attributed to the SRS-active vibration modes of these crystals.

Diode-pumped self-Q-switched single-frequency 946-nm Nd(3+), Cr(4+):YAG microchip laser.

A stable low-threshold self-Q-switched diode-pumped 946-nm Nd(3+), Cr(4+):YAG microchip laser operating in single frequency and fundamental transverse mode is reported. The output characteristics of the microchip laser were investigated under cw and pulsed pumping. By combination of the infrared microchip and an external frequency doubler, 473-nm pulses with a conversion efficiency of 18% were achieved.

Holographic reflection gratings in azobenzene polymers.

Writing and reading of reflection gratings in films of an azo side-group polymer are reported. The gratings were induced holographically by use of an argon-ion laser at 488 nm. The measured diffraction efficiency was in the range 2-10%. To estimate the scattering of the reflected light within the material, we further characterized the gratings by calculating a characteristic transmission. A distinct minimum for this transmission was observed, which was redshifted farther from the writing wavelength for gratings written in 50-microm polymer samples.

Efficient blue cw Nd:YAG microchip laser with two intracavity frequency doublers.

A microchip cavity including a half-monolithic Nd:YAG- KNbO(3) chip and a second KNbO(3) crystal is presented. An output power of up to 30 mW at 473 nm pumped by a single stripe 1-W at 808-nm diode is achieved. The laser can be operated in either single-longitudinal or multilongitudinal mode by rotation of the second frequency-doubling crystal to 0 degrees or 90 degrees from the first one. The cw output noise is less than 2% for both cases.

Efficient acousto-optic Q switching of Er:YSGG lasers at 2.79-microm wavelength.

A flash-lamp-pumped Er:Cr:YSGG laser at 2.79-microm wavelength has been acousto-optically Q switched. The Q-switched pulse energy and duration depend on pump pulse level and relative Q-switching time. Limits of single-pulse operation with the given acousto-optic diffraction efficiency have been determined. Resonator length, position of the Q switch, and output mirror reflectivity have been varied to obtain high pulse energy and the shortest pulse duration in the TEM00 transverse laser mode. A maximum single-pulse energy of 27 mJ and a minimum pulse duration of 120 ns were obtained with an output mirror reflectivity of approximately 25%. The highest Q-switched single-pulse energy amounted to 52% of the free-running, fundamental mode output pulse energy.

Molecular reorientation of a nematic glass by laser-induced heat flow.

Laser-induced birefringence patterns which break the symmetry of the experimental setup have been observed in a low molar mass anisotropic glass with nematic orientational order under a polarizing microscope. It is shown that the observed spatial modulations in birefringence can be explained by laser-induced heat flow and stress, leading to molecular reorientation which is then frozen and stored in the glassy state if the optical excitation is switched off. The experimental findings can be regarded as a manifestation of heat-flow alignment caused by a spatially inhomogeneous temperature field.

Measuring the coherence function of continuous-wave lasers by a photorefractive grating method.

We demonstrate a simple, adjustment-insensitive technique for measuring the temporal coherence function of cw multimode and monomode semiconductor lasers, using two-beam coupling in photorefractive InP and CdTe crystals. The emission spectra of the diodes are measured independently. The coherence functions are also calculated from these spectra and agree with the photorefractive measurements. Coupling of two partially coherent waves in low-speed photorefractive media is described theoretically. The range of the experimental parameters in which the method of coherence measurement is correct is given.

Properties of Nd3+-doped and undoped tetragonal PbWO4, NaY(WO4)2, CaWO4, and undoped monoclinic ZnWO4 and CdWO4 as laser-active and stimulated raman scattering-active crystals.

Spectroscopic, laser, and chi((3)) nonlinear optical properties of tetragonal PbWO(4), NaY(WO(4))(2), CaWO(4), and monoclinic CdWO(4) and ZnWO(4) were investigated. Particular attention was paid to Nd(3+)-doped and undoped PbWO(4) and NaY(WO(4))(2) crystals. Their absorption and luminescence intensity characteristics, including the peak cross sections of induced transitions, were determined. Pulsed and continuous-wave lasing in the two 4F(3/2)-->4I(11/2) and 4F(3/2)-->4I(13/2) channels was excited. For these five tungstates, highly efficient (greater than 50%) multiple Stokes generation and anti-Stokes picosecond generation were achieved. All the observed scattered laser components were identified. These results were analyzed and compared with spectroscopic data from spontaneous Raman scattering. A new crystalline Raman laser based on PbWO(4) was developed for the chi((3)) conversion frequency of 1-microm pump radiation to the first Stokes emission with efficiency up to 40%. We classify all the tungstates as promising media for lasers and neodymium-doped crystals for self-stimulated Raman scattering lasers.

Photorefractive power transfer from parallel laser diode amplifiers into a single output beam.

Photorefractive BaTiO(3):Ce was used to combine coherently a signal beam and two amplified pump beams at lambda=678 nm. The master laser beam was split and then amplified in two semiconductor laser amplifiers with gain of as much as 6. The amplifiers were made from quantum-well laser chips by use of antireflection coating. 40% of the power could be transferred to the signal beam without transferring the phase distortions of the pump beams.

Quenching of chlorophyll a fluorescence in the aggregates of LHCII: steady state fluorescence and picosecond relaxation kinetics.

The protein composition, steady state and time-resolved fluorescence emission spectra were studied in solubilized and aggregated LHCII complexes, that were prepared according to two different isolation protocols: (1) by fractionation of cation-depleted thylakoid membranes using the non-ionic detergent Triton X-100 according to the procedure of Burke et al. [(1978) Arch. Biochem. Biophys. 187, 252-263] or (2) by solubilization with N-beta-dodecyl maltoside (beta-DM) of photosystem II (PSII) membrane fragments in the presence of cations [Irrgang et al. (1988) Eur. J. Biochem. 178, 207-217]. Based on the analysis of the decay-associated emission spectra measured at 10 and 80 K five long-wavelength chlorophyll species were identified in aggregated LHCII complexes. These five forms are characterized by emission maxima at 681.5, 683, 687, 695, or 702 nm. All of these forms were found in both types of LHCII preparations but the relative amounts and temperature dependency of these species were markedly different in the aggregated LHCII complexes isolated by the two procedures. It was found that these differences cannot be simply explained by effects due to using a less mild detergent as beta-DM or by an ionic influence of Ca2+. Biochemical analysis of the protein composition showed that beta-DM type LHCII consists of all the chlorophyll (Chl)binding proteins belonging to the antenna system of PSII except the CP29 type II gene product (CP29). In contrast, the Triton X-100-solubilized LHCII is highly depleted in CP26 (CP 29 type I gene product) and is contaminated by a variety of unidentified polypeptides. It is proposed that the aggregates of LHCII prepared using Triton X-100 acquire specific spectral and kinetic features due to interaction between the bulk of LHCII subunits and minor protein(s).

Fiber phase conjugators at 1064-nm, 532-nm, and 355-nm wavelengths.

Undoped multimode quartz fibers can be used as effective phase conjugators with high fidelity and reflectivity for improving the beam quality of lasers with high average power. Such phase conjugators based on stimulated Brillouin scattering (SBS) in undoped multimode quartz fibers with a core diameter of 100 microm and a length of more than 2 m were characterized at wavelengths of 1064, 532, and 355 nm. The power thresholds at these three wavelengths are 6.4, 3.3, and 3 kW, respectively, and the maximum SBS reflectivity is more than 50% in all cases, without a significant depolarization owing to the fiber. Direct measurement of the far-field fidelity, i.e., the phase-conjugated part of the reflected light in the far field at 1064 nm, results in a fidelity of more than 90%.

Effects of hydrogen/deuterium exchange on photosynthetic water cleavage in PS II core complexes from spinach.

H/D isotope exchange effects on P680+. reduction by Yz and electron abstraction from the water oxidizing complex (WOC) in redox state S3 by YZOX were analyzed in PS II core complexes from spinach by measurements of laser flash induced absorption changes at 820 nm and 355 nm. The results obtained reveal: (1) the rate of Yz oxidation by P680+. is almost independent of the substitution of exchangeable protons by deuterons; and (2) the reaction between YZOX and the WOC in S3 exhibits a kinetic H/D isotope exchange effect of similar magnitude as that recently observed in PS II membrane fragments [Renger, G., Bittner, T. and Messinger, J. (1994) Biochem. Soc. Trans. 22, 318-322]. Based on these results it is inferred that photosynthetic dioxygen formation comprises the cleavage of at least one hydrogen bond.