Linear dichroism of light harvesting bacteriochlorophyll proteins from Rhodopseudomonas sphaeroides in stretched polyvinyl alcohol films

Light-harvesting bacteriochlorophyll-protein complexes from Rhodopseudomonas sphaeroides 2.4.1 and R-26 mutant are solubilized in sodium dodecyl sulfate and imbedded in polyvinyl alcohol. Stretching induces orientation, and the linear dichroism of visible and near infrared absorption is analyzed. Based on a simple model, angles between the particle axis and the transition dipole moments are found. In the near infrared absorption band of the R-26 light-harvesting protein the dichroic ratio varies from 1.30 to 1.57. Using the absorption curves the band is resolved into two exciton components. In the visible absorption band the dichroic ratio has a constant value of 0.43 for the R-26 protein but varies with wavelength for the wild type light-harvesting protein. This variation is attributed to an additional bacteriochlorophyll not present in the R-26 protein.     

Energy transfer in an LH4-like light harvesting complex from the aerobic purple photosynthetic bacterium Roseobacter denitrificans

A peripheral light-harvesting complex from the aerobic purple bacterium Roseobacter (R.) denitrificans was purified and its photophysical properties characterized. The complex contains two types of pigments, bacteriochlorophyll (BChl) a and the carotenoid (Car) spheroidenone and possesses unique spectroscopic properties. It appears to lack the B850 bacteriochlorophyll a Q(y) band that is typical for similar light-harvesting complex 2 antennas. Circular dichroism and low temperature steady-state absorption spectroscopy revealed that the B850 band is present but is shifted significantly to shorter wavelengths and overlaps with the B800 band at room temperature. Such a spectral signature classifies this protein as a member of the light-harvesting complex 4 class of peripheral light-harvesting complexes, along with the previously known light-harvesting complex 4 from Rhodopseudomonas palustris. The influence of the spectral change on the light-harvesting ability was studied using steady-state absorption, fluorescence, circular dichroism, femtosecond and microsecond time-resolved absorption and time-resolved fluorescence spectroscopies. The results were compared to the properties of the similar (in pigment composition) light-harvesting complex 2 from aerobically grown Rhodobacter sphaeroides and are understood within the context of shared similarities and differences and the putative influence of the pigments on the protein structure and its properties.     

Fluorescence properties of the light-harvesting bacteriochlorophyll protein from Rhodopseudomonas sphaeroides R-26

The light-harvesting bacteriochlorophyll-protein (BChl-protein) from Rhodopseudomonas sphaeroides, R-26 mutant, exhibits a strong optical absorption peak near 850 nm (Q_y band) and a weaker peak at 590 nm (Q_x band). This pigment-protein appears to contain two BChl molecules per subunit, and previous circular dichroism studies indicated the presence of excitonic interactions between the BChl molecules. The complex exhibits a fluorescence maximum near 870 nm at room temperature. Excitation in the Q_y region results in polarization p values that vary only from +0.12 at 820 nm to +0.14 near 900 nm. These values are appreciably smaller than that for monomeric BChl in viscous solvents (p > 0.4). By contrast, using Q_x excitation the p value is ?0.25 for the BChl-protein complex, which is close to that observed for the BChl monomer. For the BChl-protein these polarization values do not change greatly at a temperature of 90 K; however, the Stokes' shift of the fluorescence emission increases significantly over that at room temperature.     

Picosecond study of the near infrared absorption band of hemoglobin after photolysis of carbonmonoxyhemoglobin.

Picosecond absorption spectroscopy is used to examine the position and band shape of the near infrared absorption band of hemoglobin as a function of time after the photodissociation of CO from carbonmonoxyhemoglobin. For the earliest delay time probed, 35 ps, the peak of the transient spectrum is at 765 nm, red shifted by 6 nm from that characteristic of equilibrium deoxyhemoglobin. No evolution in either the peak position or band shape is observed for time delays up to 60 ns. In addition, the position and shape of the spectrum are independent of photolysis energies ranging from 15 microJ/pulse to 150 microJ/pulse, spanning conditions under which the photon/heme ratio is varied from 0.01 to 2.0. This indicates that the geometry in the heme group is unrelaxed and that equilibration of the surrounding protein structure occurs on a time scale longer than 60 ns.     

Bacteriochlorophyll-protein complexes of aerobic bacteria,Erythrobacter longus and Erythrobacter species OCh 114

Bacteriochlorophyll(Bchl)-protein complexes were isolated from obligate aerobic bacteria, Erythrobacter longus and Erythrobacter species OCh 114. The apparent molecular weights, absorption spectra and polypeptide compositions of the light-harvesting complexes were, in general, similar to those of the light-harvesting Bchl-protein complexes of purple photosynthetic bacteria. The reaction center complexes of these bacteria also showed similar properties to those of the purple bacteria except for slightly altered polypeptides. However, the following characteristic features of the light-harvesting systems were found in these aerobic bacteria. Major carotenoids were not bound to the Bchl-protein complex in E. longus. In Erythrobacter sp. OCh 114, a new type of Bchl-protein complex which showed a single absorption band in the near infrared region at 806 nm was obtained. The reaction center of strain OCh 114 was associated with a c-type cytochrome.Abbreviations Bchl bacteriochlorophyll a - RC reaction center - SDS sodium dodecylsulfate - PAGE polyacrylamide gel electrophoresis     

Photoselection and circular dichroism in the purple membrane.

The transient dichroic ratio D = delta A parallel/delta A perpendicular has been measured in the visible absorption region of bacteriorhodopsin in purple membrane by a flash photolysis method. D is found to be wavelength independent throughout the visible absorption band, and reaches a maximum value of 2.75 +/- 0.15 on reduction of the excitation intensity. This value is close to that expected for a single nondegenerate transition dipole moment and is incompatible with the strong exciton coupling model used to explain circular dichroism (CD) spectrum of purple membrane. A time-dependent analysis of the exciton interaction and consideration of the coupling strength suggests an explanation of these observations. It is concluded that excitation interaction between retinals in purple membrane is of the weak or very weak type defined by Förster.     

Light-induced red shift of the Qx absorption band of light-harvesting bacteriochlorophyll in Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides

In chromatophores from Rhodopseudomonas sphaeroides and Rhodopseudomonas capsulata, the Q_x band(s) of the light-harvesting bacteriochlorophyll (BChl) (λ_max ~590 nm) shifts to the red in response to a light-induced membrane potential, as indicated by the characteristics of the light-minus-dark difference spectrum. In green strains, containing light-harvesting complexes I and II, and one or more of neurosporene, methoxyneurosporene, and hydroxyneurosporene as carotenoids, the absorption changes due to the BChl and carotenoid responses to membrane potential in the spectral region 540–610 nm are comparable in magnitude and overlap with cytochrome and reaction center absorption changes in coupled chromatophores. In strains lacking carotenoid and light-harvesting complex II, the BChl shift absorption change is relatively smaller, due in part to the lower BChl/reaction center ratio.In the carotenoid-containing strains, the peak-to-trough absorption change in the BChl difference spectrum is 5–8% of the peak-to-trough change due to the shift of the longest-wavelength carotenoid band, although the absorption of the BChl band is 25–40% of that of the carotenoid band. The responding BChl band(s) does not appear to be significantly red-shifted in the dark in comparison to the total BChl Q_x band absorption.     

Circular dichroism studies of the structure of DNA complex with berberine.

The binding of the benzodioxolo-benzoquinolizine alkaloid, berberine chloride to natural and synthetic DNAs has been studied by intrinsic and extrinsic circular dichroic measurements. Binding of berberine causes changes in the circular dichroism spectrum of DNA as shown by the increase of molar ellipticity of the 270nm band, but with very little change of the 240nm band. The molar ellipticity at the saturation depends strongly on the base composition of DNA and also on salt concentration, but always larger for the AT rich DNA than the GC rich DNA. The features in the circular dichroic spectral changes of berberine-synthetic DNA complexes were similar to that of native DNA, but depends on the sequence of base pairs. On binding to DNA and polynucleotides, the alkaloid becomes optically active. The extrinsic circular dichroism developed in the visible absorption region (300-500nm) for the berberine-DNA complexes shows two broad spectral bands in the regions 425-440nm and 340-360nm with the maximum varying depending on base composition and sequence of DNA. While the 425nm band shows less variation on the binding ratio, the 360nm band is remarkably dependent on the DNA/alkaloid ratio. The generation of the alkaloid associated extrinsic circular dichroic bands is not dependent on the base composition or sequence of base pairs, but the nature and magnitude of the bands are very much dependent on these two factors and also on the salt concentration. The interpretation of the results with respect to the modes of the alkaloid binding to DNA are presented.     

Circular Dichroism Studies of the Structure of DNA Complex with Berberine

Abstract

The binding of the benzodioxolo-benzoquinolizine alkaloid, berberine chloride to natural and synthetic DNAs has been studied by intrinsic and extrinsic circular dichroic measurements. Binding of berberine causes changes in the circular dichroism spectrum of DNA as shown by the increase of molar ellipticity of the 270nm band, but with very little change of the 240nm band. The molar ellipticity at the saturation depends strongly on the base composition of DNA and also on salt concentration, but always larger for the AT rich DNA than the GC rich DNA The features in the circular dichroic spectral changes of berberine-synthetic DNA complexes were similar to that of native DNA but depends on the sequence of base pairs.

On binding to DNA and polynucleotides, the alkaloid becomes optically active. The extrinsic circular dichroism developed in the visible absorption region (300–500nm) for the berberine-DNA complexes shows two broad spectral bands in the regions 425–440nm and 340–360nm with the maximum varying depending on base composition and sequence of DNA While the 425nm band shows less variation on the binding ratio, the 360nm band is remarkably dependent on the DNA/alkaloid ratio. The generation of the alkaloid associated extrinsic circular dichroic bands is not dependent on the base composition or sequence of base pairs, but the nature and magnitude of the bands are very much dependent on these two factors and also on the salt concentration. The interpretation of the results with respect to the modes of the alkaloid binding to DNA are presented.     

Dichroic ratios in polarized Fourier transform infrared for nonaxial symmetry of beta-sheet structures

The transition moments for the amide bands from beta-sheet peptide structures generally do not exhibit axial symmetry about the director in linearly polarized Fourier transform infrared (FTIR) measurements on oriented systems. The angular dependences of the dichroic ratios of the amide bands are derived for beta-sheet structures in attenuated total reflection (ATR) and polarized transmission experiments on samples that are oriented with respect to the normal to the substrate and are randomly distributed with respect to the azimuthal angle in the plane of the orienting substrate. The orientational distributions of both the beta-strands and the beta-sheets are considered, and explicit expressions are given for the dichroic ratios of the amide I and amide II bands. The dichroic ratio of the amide II band, which is parallel polarized, can yield the orientation of the beta-strands directly, but to specify the orientations of the beta-sheets completely requires measurement of the dichroic ratios of both the amide I and amide II bands, or generally two bands with parallel and perpendicular polarizations. A random distribution in tilt of the planes of the beta-sheets does not give rise to equal dichroic ratios for bands with perpendicular and parallel polarizations, such as the amide I and amide II bands. The results are applied to previous ATR and polarized transmission FTIR measurements on a potassium channel-associated peptide, the Escherichia coli outer membrane protein OmpA, and the E. coli OmpF porin protein in oriented membranes.     

Carotenoids are essential for normal levels of dimerisation of the RC-LH1-PufX core complex of Rhodobacter sphaeroides: characterisation of R-26 as a crtB (phytoene synthase) mutant

Carotenoids play important roles in photosynthesis where they are involved in light-harvesting, in photo-protection and in the assembly and structural stability of light-harvesting and reaction centre complexes. In order to examine the effects of carotenoids on the oligomeric state of the reaction centre-light-harvesting 1 -PufX (RC-LH1-PufX) core complex of Rhodobacter sphaeroides two carotenoid-less mutants, TC70 and R-26, were studied. Detergent fractionation showed that in the absence of carotenoids LH2 complexes do not assemble, as expected, but also that core complexes are predominantly found as monomers, although levels of the PufX polypeptide appeared to be unaffected. Analysis of R-26 membranes by electron microscopy and atomic force microscopy reveals arrays of hexagonally packed monomeric RC-LH1-PufX complexes. Transfer of the crtB gene encoding phytoene synthase to TC70 and R-26 restores the normal synthesis of carotenoids demonstrating that the R-26 mutant of Rba. sphaeroides harbours a mutation in crtB, among its other defects. The transconjugant TC70 and R-26 strains containing crtB had regained their ability to assemble wild-type levels of dimeric RC-LH1-PufX core complexes and normal energy transfer pathways were restored, demonstrating that carotenoids are essential for the normal assembly and function of both the LH2 and RC-LH1-PufX complexes in this bacterial photosystem.     

Structure and fluctuation of a Streptomyces subtilisin inhibitor.

The kinetics of the hydrogen-deuterium exchange reaction in a subtilisin inhibitor from Streptomyces albogriseolus has been examined by infrared absorption measurement in aqueous solutions at various pH values and temperatures. In the analysis of each piece of kinetic data, it was assumed that the total 104 peptide hydrogen atoms are classified into three kinetic classes A, B1, and B2, and that the sizes of these classes are 72, 15, and 17, respectively at every pH and at every temperature examined. On the basis of the peak position determined for the amide II band in each stage of the exchange reaction, an approximate assignment was suggested of the A, B1 and B2 respectively to an unordered structure, a beta-structure,and an alpha-helical structure in the molecule. This assignment was supported by infrared absorption measurement of a film of this protein and by circular dichroic study of the solutions. On the basis of the temperature effect on the hydrogen-exchange rate constants and on the basis of ultraviolet absorption study in the higher temperature region (40 to 90 degrees C), a discussion has been made on the nature of the fluctuation of the molecular structure of this protein.     

Linear-dichroic triplet-minus-singlet absorbance difference spectra of reaction centers of the photosynthetic bacteria Chromatium vinosum,Rhodopseudomonas sphaeroides R-26 and Rhodospirillum rubrum S1

For the first time, linear-dichroic triplet-minus-singlet (LD-(T - S)) spectra of reaction centers of the photosynthetic bacteria Chromatium vinosum, Rhodopseudomonas sphaeroides R-26 and Rhodospirillum rubrum S1 have been measured using an extension of the technique of absorbance-detected magnetic resonance (ADMR) of the triplet state. For all bacteria studied the LD-(T - S) spectra exhibit a bleaching of the long-wavelength absorbance band that is either split or has a clear shoulder to longer wavelengths. The components are approximately parallel-polarized, indicating that they do not form an exciton pair. Around 800 nm a band appears with a width of about 7 nm, which does not form part of a band shift and that may be attributed to an appearing monomer band. Small features in the LD-(T - S) spectra at both sides of this band are well explained by band shifts of the two components of the 800 nm reaction center absorption band. The transition moment of the component at about 818 nm in reaction centers of Rps. sphaeroides R-26 is at an angle larger than 55° with both the x and the y triplet spin axes. In none of the bacteria do we find evidence for the bleaching of an exciton component of P-860 near 810 nm.     

FTIR spectroscopy of the reaction center of Chloroflexus aurantiacus: photoreduction of the bacteriopheophytin electron acceptor

Mid-infrared spectral changes associated with the photoreduction of the bacteriopheophytin electron acceptor H(A) in reaction centers (RCs) of the filamentous anoxygenic phototrophic bacterium Chloroflexus (Cfl.) aurantiacus are examined by light-induced Fourier transform infrared (FTIR) spectroscopy. The light-induced H(A)(-)/H(A) FTIR (1800-1200cm(-1)) difference spectrum of Cfl. aurantiacus RCs is compared to that of the previously well characterized purple bacterium Rhodobacter (Rba.) sphaeroides RCs. The most notable feature is that the large negative IR band at 1674cm(-1) in Rba. sphaeroides R-26, attributable to the loss of the absorption of the 13(1)-keto carbonyl of H(A) upon the radical anion H(A)(-) formation, exhibits only a very minor upshift to 1675cm(-1) in Cfl. aurantiacus. In contrast, the absorption band of the 131-keto C=O of H(A)(-) is strongly upshifted in the spectrum of Cfl. aurantiacus compared to that of Rba. sphaeroides (from 1588 to 1623cm(-1)). The data are discussed in terms of: (i) replacing the glutamic acid at L104 in Rba. sphaeroides R-26 RCs by a weaker hydrogen bond donor, a glutamine, at the equivalent position L143 in Cfl. aurantiacus RCs; (ii) a strengthening of the hydrogen-bonding interaction of the 131-keto C=O of H(A) with Glu L104 and Gln L143 upon H(A)(-) formation and (iii) a possible influence of the protein dielectric environment on the 131-keto C=O stretching frequency of neutral H(A). A conformational heterogeneity of the 133-ester C=O group of H(A) is detected for Cfl. aurantiacus RCs similar to what has been previously described for purple bacterial RCs.     

Circular dichroism of soybean leghemoglobin.

Circular dichroic (CD) spectra of soybean leghemoglobin, and some of its liganded derivatives were measured over the wavelength range of 650 to 200 nm. The heme-related circular dichroic bands in the visible, Soret and ultraviolet wavelength regions exhibit Cotton effects characteristic of each of the compounds examined. The positions of the dichroic bands vary with ligand substitutions and the oxidation state of the iron. All leghemoglobin derivatives, except the apoprotein, exhibit negative circular dichroic bands in the region of Soret absorption. In this region the optical activity of compounds with high-spin moments is greater than that of compounds with low or intermediate spin moments. The ellipticity of the heme band at about 260 nm is also altered by ligand binding and spin state. The dichroic spectra in the far-ultraviolet region indicated a high extent of alpha-helical structure (about 70%) in the native leghemoglobin and its liganded derivatives. The helicality of the apoprotein seems to diminish suggesting a decrease caused by the removal of the heme.     

Preparation and spectroscopic studies of cobalt(II)-substituted cucumber basic blue protein "plantacyanin"

The cobalt(II) derivative of cucumber basic blue copper protein "plantacyanin" has been prepared. The visible absorption, circular dichroic and magnetic circular dichroic spectra of Co(II)-plantacyanin are similar to those of Co(II)-plastocyanin, indicating that the stereochemistry of Co(II) is tetrahedral and at least one cysteinyl ligand around Co(II) ion is responsible for the strong charge transfer bands at 331 and ca. 390 nm.     

On the various types of circular dichroism induced on acridine orange bound to poly(S-carboxymethyl-L-cysteine).

Circular dichroism and absorption spectra are measured on mixed solutions of acridine orange and poly(S-carboxymethyl-L -cysteine) at different pH and P/D mixing ratios. The observed circular dichroism spectra are classified into several types, mainly based on the number and sign of circular dichroic bands in the visible region. Three of them are associated with the absorption spectra characteristic of dimeric dye or higher aggregates of dye. Type I is observed with solutions, of which the pH is acid and P/D is higher than 4, and it has an unsymmetrical pair of positive and negative dichroic bands at 470 and 430 nm. This type is induced on the dye bound to the polymer in the β-conformation. Types II and III are considered to be characteristic of randomly coiled polymers. Type II is exhibited by solutions of P/D higher than 1 at pH 5–7 and has two dichroic bands around the same wavelengths as Type I but with opposite signs and an additional positive band at 560 nm. Type III, shown by solutions of P/D 2–0.6 at pH 6–10.5, has three dichroic bands around the same wavelengths as Type II but with signs opposite to it. The other two types of circular dichroism, induced for the solutions of P/D less than 1 at slightly acid pH, are associated with the absorption spectra of monomeric dye and are observed with disordered or randomly coiled polymer. They have a pair of dichroic bands at 540 and 425 nm, and the signs of these bands are opposite to each other in these two types.     

Surface Plasmon Resonance Study of Au Nanorod Structures Templated in Mesoporous Silicon

Au nanorods forming carpet-like structures have been fabricated by electrochemical methods in mesoporous silicon templates. The anodically grown monodisperse pores on Si (100) were used to cathodically grow the Au structures from complexated Au solutions. Such structures feature selective absorption of light in the visible/near infrared wavelength range that can be tuned by controlling the aspect ratio and dielectric surrounding media. In the present work, the visible light absorption of Au structures has been studied and correlated with the light absorption of ideal structures with increasing complexity. According to these simulations, the observed selective light absorption arises from the anisotropic surface plasmon resonance (SPR) of the rods and a secondary SPR of isolated Au nanoparticles adsorbed on the rods.     

Conformational changes in plastocyanin

The visible and near-uv absorption and circular dichroic spectra were determined for spinach and poplar plastocyanin under a variety of conditions. The visible spectra showed that the copper center was invariant to changes in species, chemical modification with ethylenediamine, and addition of high concentrations of salt [2.7 M (NH4)2SO4]. In contrast, the near-uv spectra were sensitive to these conditions. Reduction of plastocyanin also altered its near-uv absorption and circular dichroic spectra. It is unlikely that these spectral changes were due to charge transfer bands since the near-uv CD spectrum of apo-plastocyanin was almost identical to that of reduced plastocyanin. There were no corresponding changes in the far-uv spectra which monitor protein secondary structure. The most likely explanation is that the protein has a flexible tertiary conformation. Conformational changes may be important in regulating electron transport. If plastocyanin is a mobile electron carrier, differential binding of the oxidized and reduced forms of plastocyanin to its reaction partners cytochrome f and P700 could facilitate electron transport.     

Spectroscopic characterization of the spinach Lhcb4 protein (CP29), a minor light-harvesting complex of photosystem II.

A spectroscopic characterization is presented of the minor photosystem II chlorophyll a/b-binding protein CP29 (or the Lhcb4 protein) from spinach, prepared by a modified form of a published protocol [Henrysson, T., Schroder, W. P., Spangfort, M. & Akerlund, H.-E. (1989) Biochim. Biophys. Acta 977, 301-308]. The isolation procedure represents a quicker, cheaper means of isolating this minor antenna protein to an equally high level of purity to that published previously. The pigment-binding protein shows similarities to other related light-harvesting complexes (LHCs), including the bulk complex LHCIIb but more particularly another minor antenna protein CP26 (Lhcb5). It is also, in the main, similar to other preparations of CP29, although some significant differences are discussed. In common with CP26, the protein binds about six chlorophyll a and two chlorophyll b molecules. Two chlorophyll b absorption bands are present at 638 and 650 nm and they are somewhat more pronounced than in a recent report [Giuffra, E., Zucchelli, G., Sandonà, D., Croce, R., Cugini, D., Garlaschi, F.M., Bassi, R. & Jennings, R.C. (1997) Biochem. 36, 12984-12993]. The bands give rise to positive and negative linear dichroism, respectively; both show negative CD bands (cf. bands with similar properties at 637 and 650 nm in CP26). Chlorophyll a absorption is dominated by a large contribution at 674 nm which also shows similarities to the major band in LHCIIb and CP26, while (as for CP26) a reduction in absorption around 670 nm is observed relative to the bulk complex. Principal differences from LHCIIb and CP26, and from other CP29 preparations, occur in the carotenoid region.