Effect of 13-cis violaxanthin on organization of light harvesting complex II in monomolecular layers

Lutein, neoxanthin and violaxanthin are the main xanthophyll pigment constituents of the largest light-harvesting pigment-protein complex of photosystem II (LHCII). High performance liquid chromatography analysis revealed photoisomerization of LHCII-bound violaxanthin from the conformation all-trans to the conformation 13-cis and 9-cis. Maximally, the conversion of 15% of all-trans violaxanthin to a cis form could be achieved owing to the light-driven reactions. The reactions were dark-reversible. The all-trans to cis isomerization was found to be driven by blue light, absorbed by chlorophylls and carotenoids, as well as by red light, absorbed exclusively by chlorophyll pigments. This suggests that the photoisomerization is a carotenoid triplet-sensitized reaction. The monomolecular layer technique was applied to study the effect of the 13-cis conformer of violaxanthin and its de-epoxidized form, zeaxanthin, on the organization of LHCII as compared to the all-trans stereoisomers. The specific molecular areas of LHCII in the two-component system composed of protein and exogenous 13-cis violaxanthin or 13-cis zeaxanthin show overadditivity, which is an indication of the xanthophyll-induced disassembly of the aggregated forms of the protein. Such an effect was not observed in the monomolecular layers of LHCII containing all-trans conformers of violaxanthin and zeaxanthin. 77 K chlorophyll a fluorescence emission spectra recorded from the Langmuir-Blodgett (L-B) films deposited to quartz from monomolecular layers formed with LHCII and LHCII in the two-component systems with all-trans and 13-cis isomers of violaxanthin and zeaxanthin revealed opposite effects of both conformers on the aggregation of the protein. The cis isomers of both xanthophylls were found to decrease the aggregation level of LHCII and the all-trans isomers increased the aggregation level. The calculated efficiency of excitation energy transfer to chlorophyll a from violaxanthin assumed to remain in two steric conformations was analyzed on the basis of the chlorophyll a fluorescence excitation spectra and the mean orientation of violaxanthin molecules in LHCII (71 degrees with respect to the normal to the membrane), determined recently in the linear dichroism experiments [Gruszecki et al., Biochim. Biophys. Acta 1412 (1999) 173-183]. The calculated efficiency of excitation energy transfer from the violaxanthin pool assumed to remain in conformation all-trans was found to be almost independent on the orientation angle within a variability range. In contrast the calculated efficiency of energy transfer from the form cis was found to be strongly dependent on the orientation and varied between 1.0 (at 67.48 degrees ) and 0 (at 70.89 degrees ). This is consistent with two essentially different, possible functions of the cis forms of violaxanthin: as a highly efficient excitation donor (and possibly energy transmitter between other chromophores) or purely as a LHCII structure modifier.     

Magnetic circular dichroism of netropsin and natural circular dichroism of the netropsin-DNA complex

We report the first measurement of the magnetic circular dichroism (MCD) of the basic polypeptide antibiotic netropsin (Nt). The MCD shows that the longest wavelength absorption band of Nt is the sum of more than one component and permits a radically new interpretation of the circular dichroism of the complex which Nt forms with DNA. We conclude that Nt has no major effect on the CD and thus the helical structure of the bases of the DNA to which it is bound. Thus the ability of Nt to inhibit the function of DNA polymerase, RNA polymerase, and the photoreactivating enzyme must be mediated by factors other than a distortion of the helical structure of the bases.     

Structural-functional organization of the main light harvesting complex and photosystem 2 of higher plants

The interrelation between spectral and structural–functional properties of LhcIIb was studied. The dipole strength of the main Q_y bands of chlorophylls (Chl a 30.8 D²; Chl b 18.5 D²) and chlorophyll a/b ratio (Chl a/Chl b = 7 : 6) were determined for LhcIIb. The Chl a/Chl b value shows that the subunit of this complex contains seven Chl a and six Chl b molecules. Individual bands of chlorophylls (bands in stokes and anti-Stokes parts at 77 K were Lorentzian and Gaussian, respectively) were resolved using synchronized deconvolution of absorption, CD, and LD bands of chlorophylls. Seven of these bands belonged to Chl a. Parameters of absorption bands of Chl a indicate that seven molecules represent a united cluster (heptamer) with exciton interactions, determining the spectrum of LhcIIb in the Chl a absorption region. Parameters of absorption bands of Chl b show the existence of three clusters: monomer (639.6 nm), dimer (645.2 and 647.4 nm), and trimer (649.8 and 654.1 nm). These clusters and their properties agree with the well-known structure of porphyrin groups of the LhcIIb subunit (Kuhlbrandt, 1994). A distorted ring of seven porphyrins in the stromal range of the subunit corresponds to Chl a heptamer; a separately located molecule near the N-terminal domain on the stromal side of the subunit corresponds to Chl b monomer; a dimer and a trimer of porphyrins in the lumenal range of the subunit correspond to the dimer and trimer of Chl b, respectively. The calculated lifetimes of the excitation energy (exciton) transfer in subunit and trimer of LhcIIb confirm this location of pigments. The geometry of the Chl a heptamer (mutual orientation of transition dipole moments) was determined by the steady-state Kasha–Tinoco approximation using parameters of individual bands of exciton splitting. The calculated parameters of mutual orientation of Chl a dipoles agree with the topography of the stromal porphyrins found by electron crystallography (Kuhlbrandt, 1994). A structural model of the granal multicentral macrocomplex of PSII (MPSII) is suggested. The lifetimes of the exciton migration between the main pigment–protein compartments of MPSII were calculated. The results of calculation are consistent with the structural model of the photosystem. The location of pigments provides for fast exciton hopping between Chl a clusters of neighboring proteins in the MPSII along the stromal surface within the membrane (5-25 psec) and between stacked membranes (40 psec) of chloroplast grana.     

Calculation of spectra of absorption and circular dichroism of Rhodopseudomonas acidophila light harvesting complexes based on roentgen structural data

Absorption and circular dichroism spectra of two forms of the peripheral light-harvesting complex from photosynthetic purple bacteria Rhodopseudomonas acidophila were calculated. Calculations were carried out on the basis of exciton theory for circular aggregates of bacteriochlorophyll molecules and X-ray data for these forms of the complex. It was shown that theoretical spectra fit well experimental ones at the same values of excitation energy, homogeneous and inhomogeneous broadening, and bandwidth for all bacteriochlorophyll molecules of complexes. To approximate the circular dichroism spectra of complexes, it was necessary to change the orientations and the values of the moments of transition of Qy molecules relative to their orientation determined on the basis of X-ray structure analysis data.     

Seasonal changes in photosystem II organisation and pigment composition in Pinus sylvestris

Conifers of the boreal zone encounter considerable combined stress of low temperature and high light during winter, when photosynthetic consumption of excitation energy is blocked. In the evergreen Pinus sylvestris L. these stresses coincided with major seasonal changes in photosystem II (PSII) organisation and pigment composition. The earliest changes occurred in September, before any freezing stress, with initial losses of chlorophyll, the D1-protein of the PSII reaction centre and of PSII light-harvesting-complex (LHC II) proteins. In October there was a transient increase in F₀, resulting from detachment of the light-harvesting antennae as reaction centres lost D1. The D1-protein content eventually decreased to 90%, reaching a minimum by December, but PSII photochemical efficiency [variable fluorescence (F_v)/maximum fluorescence (F_m)] did not reach the winter minimum until mid-February. The carotenoid composition varied seasonally with a twofold increase in lutein and the carotenoids of the xanthophyll cycle during winter, while the epoxidation state of the xanthophylls decreased from 0.9 to 0.1 from October to January. The loss of chlorophyll was complete by October and during winter much of the remaining chlorophyll was reorganised in aggregates of specific polypeptide composition, which apparently efficiently quench excitation energy through non-radiative dissipation. The timing of the autumn and winter changes indicated that xanthophyll de-epoxidation correlates with winter quenching of chlorophyll fluorescence while the drop in photochemical efficiency relates more to loss of D1-protein. In April and May recovery of the photochemistry of PSII, protein synthesis, pigment rearrangements and zeaxanthin epoxidation occurred concomitantly. Indoor recovery of photosynthesis in winter-stressed branches under favourable conditions was completed within 3 d, with rapid increases in F₀, the epoxidation state of the xanthophylls and in light-harvesting polypeptides, followed by recovery of D1-protein content and F_v/F_m, all without net increase in chlorophyll. The fall and winter reorganisation allow Pinus sylvestris to maintain a large stock of chlorophyll in a quenched, photoprotected state, allowing rapid recovery of photosynthesis in spring.Abbreviations Elips early light-induced proteins - EPS epoxidation state - F₀ instantaneous fluorescence - F_m maximum fluorescence - F_v variable fluorescence - LHC II light-harvesting complex of PSII - LiDS lithium dodecyl sulfate This research was supported by the Swedish Natural Science Research Council. We wish to thank Dr. Adrian Clarke¹ (Department of Plant Physiology, University of Umeå, Sweden) for advice on electrophoresis, valuable discussion and providing antibodies. Dr. Stefan Jansson¹ and Dr. Torill Hundal (Department for Biochemistry, University of Stockholm, Sweden) provided antibodies. Jan Karlsson¹ helped with the HPLC, Dr. Marianna Krol gave advice on green gels and Dr. Vaughan Hurry (Cooperative Research Centre for Plant Sciences, Australian National University, Canberra, Australia) provided valuable discussion.     

Collagenase induced changes in the circular dichroism spectrum of collagen

The maximum at 220 nm in the circular dichroism spectrum of native collagen solution changed to a negative value after heat denaturation or collagenase hydrolysis. The enzyme induced rate of CD change at 220 nm was shown to be first order in collagen concentration. The specific rate constant k is actually a combined rate constant k_fast and k_slow in which the ratio k_f/k_s is 4.1. The initial rates were linear with respect to enzyme concentration, and the K_m was found to be 5.5 × 10^?7 M. The rate of ultraviolet hyperchromicity at 220 nm on collagen hydrolysis was determined. The k_fast was the same as that obtained by CD. The k_f/k_s ratio was 4.6. Both methods may be readily used to assay for collagenase activity.     

Model for fluorescence quenching in light harvesting complex II in different aggregation states

Low-temperature (77 K) steady-state fluorescence emission spectroscopy and dynamic light scattering were applied to the main chlorophyll a/b protein light harvesting complex of photosystem II (LHC II) in different aggregation states to elucidate the mechanism of fluorescence quenching within LHC II oligomers. Evidences presented that LHC II oligomers are heterogeneous and consist of large and small particles with different fluorescence yield. At intermediate detergent concentrations the mean size of the small particles is similar to that of trimers, while the size of large particles is comparable to that of aggregated trimers without added detergent. It is suggested that in small particles and trimers the emitter is monomeric chlorophyll, whereas in large aggregates there is also another emitter, which is a poorly fluorescing chlorophyll associate. A model, describing populations of antenna chlorophyll molecules in small and large aggregates in their ground and first singlet excited states, is considered. The model enables us to obtain the ratio of the singlet excited-state lifetimes in small and large particles, the relative amount of chlorophyll molecules in large particles, and the amount of quenchers as a function of the degree of aggregation. These dependencies reveal that the quenching of the chl a fluorescence upon aggregation is due to the formation of large aggregates and the increasing of the amount of chlorophyll molecules forming these aggregates. As a consequence, the amount of quenchers, located in large aggregates, is increased, and their singlet excited-state lifetimes steeply decrease.     

Conformational changes in liver alcohol dehydrogenase upon nucleotide binding: detection by circular dichroism of dye complexes

Michler's ketone, the ketone analog of auramine O, binds to liver alcohol dehydrogenase with an affinity comparable to that of auramine O, yields similar induced circular dichroism bands, and its binding is enhanced in the same way by binding of coenzyme fragments. These observations suggest that (a) Michler's ketone binds to liver alcohol dehydrogenase in a similar manner and at the same site as does auramine O and (b) the enhancement of auramine O and Michler's ketone binding by coenzyme fragments is not due to electrostatic interactions since both the positively charged auramine O and the neutral Michler's ketone show similar effects. Observation of similar enhancement of auramine O binding by GMP and ?-AMP as well as the activesite topology suggested by fluorescence and X-ray studies argue against any direct interaction of the dye with the purine ring. We, therefore, ascribe the enhanced binding of dye in ternary complexes to a conformational change in liver alcohol dehydogenase induced by the binding of coenzyme fragments. Studies of the circular dichroism of liver alcohol dehydrogenase complexes with 2,2′-bipyridyl are also reported.     

Circular dichroism and magnetic circular dichroism spectra of chlorophylls a and b in nematic liquid crystals. II. Magnetic circular dichroism spectra

Absorption and magnetic circular dichroism (MCD) spectra are reported for chlorophyll (Chl) a and Chl b dissolved in nematic liquid crystal solvents. The spectra were measured with the dye molecules oriented uniaxially along the direction of. the magnetic field and measuring light beam. It is significant that under such conditions the MCD spectra recorded in the wavelength region of the Q and Soret bands of the chlorophyll are essentially unchanged with respect to rotation of the sample cell around this axis, even though there is almost complete orientation of the chlorophyll molecules by the liquid crystals. The MCD spectra of Chl a and b in the nematic liquid crystal solvents used in this study are surprisingly similar to the spectra obtained under isotropic conditions. These results illustrate an important technique with which to examine the optical spectra of dyes oriented in liquid crystal matrices in which the anisotropic effects can be reduced the negligible proportions by the application of a strong magnetic field parallel to the direction of the measuring light beam. The first deconvolution calculations are reported that describe the deconvolution of pairs of absorption and MCD spectra, in the Q and B band regions, for both Chl a and b. The spectral analysis to obtain quantitative estimates of transition energies was accomplished by carrying out detailed deconvolution calculations in which the both the absorption and MCD spectral envelopes were fitted with the same number of components; each pair of components had the same hand centres and bandwidth values. This procedure resulted in an assignment of each of the main transitions in the absorption spectra of both Chl a and b. Chl a is clearly monomeric, with Qy, Qx, By and Bx located at 671, 582, 439 and 431 nm, respectively. Analysis of the spectral data for Chl b located Qy, By and Bx, at 662, 476 and 464 nm, respectively.     

Study of the pyruvate dehydrogenase complex by circular dichroism]

A comparative study of the pyruvate dehydrogenase complex and its pyruvate dehydrogenase component was carried out by using the circular dichroism method. It was found that the spectral properties of the pyruvate dehydrogenase complex are determined by those of its first component: i) the spectrum of the thiamine pyrophosphate-free pyruvate dehydrogenase complex displayed the main characteristics of the pyruvate dehydrogenase component; ii) the appearance of the charge transfer complex band during thiamine pyrophosphate saturation was revealed for the both proteins; iii) in both cases the charge transfer complex band disappeared after the interaction of the holoform with pyruvate and reappeared after the addition of dithiothreitol used as a deacetylating reagent. Coenzyme A in the same reaction selectively deacetylated the pyruvate dehydrogenase complex (but not its pyruvate dehydrogenase component). The spectral dynamics of pyruvate dehydrogenase reflects the functional changes in the enzyme active centers during the catalytic act. The similarity of the spectral behaviour of pyruvate dehydrogenase within the complex structure and in the isolated state provides support for the earlier proposed mechanism of the pyruvate dehydrogenase action and ensures a methodological basis for its direct investigation within the complex structure.     

The calculated circular dichroism of polyproline II in the polarizability approximation

The circular dichroism (CD) spectrum of polyproline II (PPII) has heretofore been moderately well calculated from exciton theory only at the expense of assuming unreasonable chain conformations and accepting a conservative spectrum in the 180–250-nm region (which is not observed). We have incorporated far uv transitions in the polarizability approximation and, together with the π₂π* transition, have calculated the resulting correction to the exciton model. This has been accompanied by a modified assignment of the ππ* transition in PPII, and a simultaneous calculation of the absorption and CD spectra of the α-helix, β structure, PPI, and PPII. We obtain good agreement with the observed CD spectrum of PPII in the 180–250-nm region for acceptable chain conformations. In addition, we predict a negative CD into the far uv, in agreement with recent experimental observations. Our calculations also reproduce features of the far uv CD spectrum of the α-helix, and are in agreement with the CD spectra of the β chain and PPI. The calculated CD of the unordered polypeptide chain is not significantly influenced by far uv contributions, indicating that our previous calculation is valid for such a system. These results demonstrate the importance of incorporating far uv transitions in order to achieve an adequate theoretical explanation of the CD spectra of polypeptides.     

A Bloch equation approach to intensity dependent optical spectra of light harvesting complex II

On the basis of the recent progress in the resolution of the structure of the antenna light harvesting complex II (LHC II) of the photosystem II, we propose a microscopically motivated theory to predict excitation intensity-dependent spectra. We show that optical Bloch equations provide the means to include all 2( N ) excited states of an oligomer complex of N coupled two-level systems and analyze the effects of Pauli Blocking and exciton-exciton annihilation on pump-probe spectra. We use LHC Bloch equations for 14 Coulomb coupled two-level systems, which describe the S (0) and S (1) level of every chlorophyll molecule. All parameter introduced into the Hamiltonian are based on microscopic structure and a quantum chemical model. The derived Bloch equations describe not only linear absorption but also the intensity dependence of optical spectra in a regime where the interplay of Pauli Blocking effects as well as exciton-exciton annihilation effects are important. As an example, pump-probe spectra are discussed. The observed saturation of the spectra for high intensities can be viewed as a relaxation channel blockade on short time scales due to Pauli blocking. The theoretical investigation is useful for the interpretation of the experimental data, if the experimental conditions exceed the low intensity pump limit and effects like strong Pauli Blocking and exciton-exciton annihilation need to be considered. These effects become important when multiple excitations are generated by the pump pulse in the complex.     

On photoprotective mechanisms of carotenoids in light harvesting complex

Carotenoids in light harvesting complex (LHC) play an important role in preventing plants photodamage caused by excess light. Non-photochemical quenching (NPQ) is an important mechanism adopted by plants to deal with high light intensity and the major component is referred to as energy dependent quenching (qE). Despite numerous studies have been devoted to investigating the site and mechanism of qE, there are still much debate on these topics. In this article, we discussed the possible site and underlying mechanism of qE based on the structural similarity of carotenoids. Moreover, being as good antioxidants, carotenoids’ potential protective effects against LHC photo-oxidation by quenching active oxygen species or triplet excited state chlorophyll are also discussed.     

Improvement of microalgal photosynthetic productivity by reducing the content of light harvesting pigment

Microalgal productivity was examined using both a wild type and a phycocyanin-deficient mutant of Synechocystis PCC 6714 (PD-1). The culture was conducted at various light intensities under low and high cell densities in a continuous culture system. At low light intensity, photosynthetic productivity was almost the same for both low and high cell densities. However, at higher light intensities photosynthetic productivity was higher in mutant PD-1 than in the wild type. At 2000 μmol photon m⁻² s⁻¹ the productivity was 50% higher in mutant PD-1. This result is consistent with our first report (Nakajima & Ueda, 1997), which showed that photosynthetic productivity can be improved by reducing the light harvesting pigment content in high cell density cultures at high light intensities. It is concluded that the technology for reducing LHP content is a useful method for improving photosynthetic productivity in algal mass production. This revised version was published online in July 2006 with corrections to the Cover Date.