NEW SPECTRAL COMPONENTS IN HIGH RESOLUTION ABSORPTION SPECTRA OF GREEN BACTERIAL REACTION CENTER COMPLEXES AT 5K*

Abstract—absorption spcctra of reaction center Complexes I and II from Chlorobium limicola f. thiosul-fatophilum were taken from 760 and 860 nm at 5 K. Fourth and eighth derivatives of the spectra were calculated from the digital data. Light-minus-dark difference spectra were taken, also at 5 K, with 590 nm actinic light. A shoulder not visible at 77 K appears on the long wavelength side of the 834 nm peak in Complex I. In Complex II, which is derived by guanidine HCI treatment of I, the shoulder is much more pronounced; derivative peaks appear at 834 and 838 nm. In the difference spectra, there are troughs at 832 and 838 nm. The latter trough is the first instance in green bacteria of a wavelength coincidence between a light-induced bleaching and a peak in (derivative) absorbance. There is also a nearly symmetrical pair of features, a trough at 814 nm and a peak at 818 nm, that appear to represent a light-induced bathochromic shift of the absorbance at 816 nm, a peak which occurs in both complexes as well as the photochemically inert bacteriochlorophyll a (Bchl a) protein. Other features in the absorption spcctra of both Complexes occur at virtually the same wavelengths as the peaks in purified Bchl a-protein trimer. We conclude that a large fraction of the Bchl a in Complex II is in a conformation similar to that of a single subunit of Bchl a-protein.     

PHYCOBILIPROTEINS: COMPARISON OF SOLUTION AND SINGLE CRYSTAL FLUORESCENCE FOR C-PHYCOCYANIN AND B-PHYCOERYTHRIN*

Abstract— Trimeric and hexameric solution forms of C-phycocyanin (CPC) from the cyanophyte Agme-nellum quadruplicatum have been isolated and their spectral properties compared to those obtained from single crystals. Although the absorbance peak of a suspension of small C-phycocyanin crystals is red-shifted only 7 nm relative to the solution forms, the single crystal fluorescence is red-shifted 60 nm relative to the solution forms. The crystal fluorescence spectrum exhibits a single peak at LD_max= 708 nm when excited at 514.5 or 530.9 nm and two peaks (LD_max= 661 and 708 nm) when excitation occurs at 568.2 nm. Fluorescence depolarization measurements indicate that extensive energy transfer could occur for both solution and crystal forms with the latter being dependent upon the relative orientation of the crystal with respect to the excitation dipole. Similar results were obtained with B-phycoerythrin (BPE) from the red alga Porphyridium cruentum where the single crystal fluorescence is red-shifted =50nm relative to the solution spectra with two peaks (LD_max= 583 and 617 nm) observed whose relative intensities are dependent on the excitation wavelength (LD_max– 514.5 and 530.9 nm). Single crystal fluorescent lifetimes exhibited considerable shortening relative to that observed for the solution forms. The implications of these results are discussed with respect to the possible relationships of the crystalline structures to the assembly forms present within phycobilisomes.     

THE EFFECT OF PERIPHERAL SUBSTITUTION ON THE BATHOCHROMIC SHIFT OF THE Qy TRANSITION OF BACTERIOCHLOROPHYLL DIMERS; in vitro MODELS OF THE PROTEIN EFFECT ON THE SPECTRUM OF PIGMENT CENTERS IN THE LIGHT-HARVESTING COMPLEXES*

Abstract— The effect of chemical modifications in the side groups of the isocyclic ring V on the formation, optical absorption and circular dichroism of bacteriochlorophyll (Bchl) dimers was examined in a mixture of formamide and water containing TritonX–100 and variable amounts of pyridine. Substitution of the carbomethoxy group in the C13² position with a hydrogen atom, had no effect on the dimerization constant but increased the shift of the Q_y transition by 1000 cm^-1 with respect to the native Bchl. Substitution of the C13 hydrogen atom with OH decreased the shift of the Q_y transition by 400 cm^-1. The similarity between the spectra of the modified Bchl dimers and Bchl dimers in vivo indicates that protein binding to the side groups at Bchl dimers may profoundly affect the energy of their Q_y transition but have minor effects on the Q_y transitions of the monomelic Bchl.     

NEAR-INFRARED-FT-RAMAN STUDY OF AGGREGATION OF BACTERIOCHLOROPHYLL c IN WHOLE LIVING Chlorobium limicola

Near-infrared excited Fourier-transform Raman spectra have been measured for whole living Chlorobium limicola f. thiosulfatophilum to explore in situ structure of bacteriochlorophyll c (BChl-c). The spectra, whose Raman bands are preresonance enhanced via a Qy band of BChl-c, did not contain contributions from the major components of bacteria such as proteins and lipids. Therefore, the spectra provide selective structural information about BChl-c in the chlorosomes in a totally nondestructive manner. A marker band, appearing at 1605 cm-1 for the coordination number of the Mg atom, shows that BChl-c in the chlorosomes is five coordinate. The Raman spectrum of living bacteria closely resembles that of BChl-c in water-saturated carbon tetrachloride (w-std CC14) where it is comprised of dimer, tetramer and polymer spectra. However, a band assigned to a C=O stretching mode of the 131-keto group is identified only at 1641 cm-1. A band arising from the free keto carbonyl group, which appears in the spectrum of BChl-c in w-std CC14, is not observed in the spectra of bacteria. These observations suggest that BChl-c in the chlorosomes forms mostly coordinate polymeric species whose structure is very similar to that of BChl-c in w-std CC14.     

OBSERVATION OF THE PICOSECOND TIME-RESOLVED SPECTRUM OF SQUID BATHORHODOPSIN AT ROOM TEMPERATURE

Difference spectra between squid rhodopsin and its bathorhodopsin at room temperature were measured ca. 150 ps and ca. 500 ps after the excitation at 347.2 nm by a double-beam picosecond time-resolved spectrometer. The spectra measured showed a red shift of the isosbestic point between squid rhodopsin and its bathorhodopsin and a lower ΔA_max/ΔA_min value compared with those measured at low temperatures by conventional spectrophotometry.     

ON THE MULTIPLE CYCLES OF BACTERIORHODOPSIN AT HIGH pH

Bacteriorhodopsin (bR) shows at least two parallel photocycles at pH 10.5, suggesting that more than one form of bR exists in alkaline bR sample. Upon the absorption of visible light, the different forms of bR at high pH yield different parallel intermediates: M₄₁₂ with two rise and two decay components; and R, an extremely fast rising and extremely slow decaying intermediate with an absorption peak at 350 nm. The kinetics and spectra do not agree with the proposal of Kouyama el al. (1988, Biochemistry 27,5855–5863) that the 350 nm-absorbing species is the N intermediate which follows M₄₁₂ and that the slow decaying M₄₁₂ is an M-like photoproduct of N. Our results basically agree with the proposal of Dacshazy et al. (1988, Proc. Natl. Acad. Sci. USA 85,6358–6361) that the fast and slow decaying M₄₁₂ intermediates and R are in independent photocycles arising from different forms of bR. The different forms of bR are probably in dynamic equilibrium with their ratios controlled by pH and ionic strength.     

THE MAJOR INTRINSIC LIGHT-HARVESTING PROTEIN OF Amphidinium: CHARACTERIZATION AND RELATION TO OTHER LIGHT-HARVESTING PROTEINS

A major light-harvesting complex (LHC) has been obtained from thylakoids of Amphidinium carterae solubilized with digitonin or decylmaltoside and separated by sucrose-gradient centrifugation. The digitonin-LHC forms a dark brown band at -17% sucrose and the decylmaltoside LHC one at -7% sucrose. Excellent energy transfer is retained from chlorophyll c and carotenoid to chlorophyll a. Absorbance and fluorescence excitation spectra show the existence of two major forms of chlorophyll c, one absorbing at 634 nm and the other at 649 nm. Linear dichroism spectra show the Q_y transition of both forms of chlorophyll c to be aligned at <35° to the membrane plane. On sodium dodecylsulfate polyacrylamide gels the complex resolves as a single band of 19 kDa. A partial amino acid sequence shows the N-terminus to be unblocked but modified; there is a persistent ambiguity of Ser/Asn at residue 4 and evidence for multiple but very similar polypeptides within the 19 kDa band. The peptide has strong identity with the N-terminal regions of LHC from Phaeodactylum and Pavlova and LHC 1 of higher plants. Antibodies to the 19 kDa peptide react weakly with LHC of brown algae, diatoms and Prymnesiophytes but not with those of higher plants or Cryptophytes.     

ACTION SPECTRA FOR LIGHT-INDUCED DE-EPOXIDATION AND EPOXIDATION OF XANTHOPHYLLS IN SPINACH LEAF*

Abstract— The action spectra for violaxanthin de-epoxidation and zeaxanthin epoxidation in New Zealand spinach leaf segments, Tetragonia expansa, were determined at equal incident quanta of 2·0 × 10¹⁵ quanta cm^-2 sec^-1. Precise action spectra were not obtained due to variable leaf activity. The de-epoxidation action spectrum had major peaks at approximately 480 and 648 nm. Blue light was slightly more effective than red light and little activity was observed beyond 700 nm. The epoxidation action spectrum showed major peaks at around 440 and 670 nm. Blue light was more effective than red light and light beyond 700 nm showed definite activity. The net result of de-epoxidation and epoxidation is a cyclic scheme, the violaxanthin cycle, which consumes O₂ and photoproducts. The action spectra indicate that the violaxanthin cycle is more active in blue than in red light and therefore could account for O₂ uptake stimulated by blue light. However, the violaxanthin cycle is not the pathway for O₂ uptake by photosynthetic system 1. It was suggested that the violaxanthin cycle may function as a pathway for the consumption of excess photoproducts generated in blue light or the conversion of these photo-products to other forms of energy.     

A MODEL FOR THE MOLECULAR STRUCTURE AND ORIENTATION OF THE CHROMOPHORE IN A DIMERIC PHYTOCHROME MOLECULE*

A model for the molecular structure and orientation of red-light absorbing form of phytochrome (P,) chromophores in a dimeric molecular model of P_r is proposed. A chromophore model with probable molecular structures was generated to reproduce the absorption spectrum produced by its π-electron conjugating system. The model has C₅-Z, syn, C₁₀-E, anti and C₁₅-Z, syn configurations and a protonation at a C-ring nitrogen. Orientation of the chromophore model in the dimeric phytochrome molecular was analyzed by displaying the atoms of the chromophore, the coordinates of which were converted into those with respect to the molecular axes to the dimeric molecule, on a 3-D graphic workstation. The conversions were performed by using the azimuthal angles between the Z axis of the dimeric molecule (axis of 2-fold rotational symmetry) and the dipole moments of the electronic transition at the blue- (384 nm) and red- (667 nm) absorbing bands of the chromophore, which were calculated as 55.5° and 59.3°, respectively, based on linear dichroism of the oriented phytochrome molecules. The result demonstrates that the long axis of the P, chromophore lies almost parallel to the Y axis of the molecular model, and that the tetrapyrrolic chromophore is well contained within the flat chromophoric domain without protruding from it, a configuration that assures that the chromophore is protected against aqueous environments. The model may explain the rotation angle of the transition moment of the red-absorbing band, induced by the phototransformation from P_r to P_rr which we measured as smaller than that measured in nonoriented preparations by a photoselection technique. The model also suggests a molecular basis for the polarotropic response of phytochrome.     

Zur Deutung des UV.-Spektrums von Pyridin-N-oxid

From measurements of the influence of an electric field on the absorption spectrum of pyridine-N-oxide it is concluded that the 330 nm band is polarized perpendicular to the dipole moment, while the 280 nm transition moment lies parallel. Furthermore from these experiments the dipole moments in both excited states have been determined (Table 1). PARISER -PARR -POPLE -calculations as well as CNDO-calculations admit an assignment of the 330 nm band to an A₁ → B₁, π → π* transition and of the 280 nm band to an A₁ → A₁, π → π* transition. Thereby energy, polarization, intensity of the transition, and the dipole moments of the excited states have been taken into consideration. This assignment does not exclude the possibility of a weak n-π* transition at approximately the same wavelength as the A₁ → B₁ transition.     

AQUEOUS AGGREGATES OF BACTERIOCHLOROPHYLL c AS A MODEL FOR PIGMENT ORGANIZATION IN CHLOROSOMES

Chlorosomes isolated from Chloroflexus aurantiacus were extracted with chloroform/methanol. The extract contained bacteriochlorophylls c and a and lipids, but was devoid of proteins. This crude extract spontaneously formed aggregates when a methanol solution was dispersed in aqueous buffer. The aggregates could be sedimented by ultracentrifugation and appeared in electron micrographs as stain-excluding bodies with diameters between 70 and 170 nm. The absorption spectrum is remarkably similar to that of intact chlorosomes with an absorption maximum of bacteriochlorophyll c at around 740 nm. The circular dichroism spectrum of the aggregate is also very similar to that of intact chlorosomes. A conservative (±) band centered at 740 nm confirms the highly aggregated state of bacteriochlorophyll c in both systems. Steady-state fluorescence studies showed that in the aggregate energy-transfer from bacteriochlorophyll c to a component emitting at 830 nm took place. When the aggregate was suspended in buffer saturated with 1-hexanol the 740 nm form of bacteriochlorophyll c was reversibly converted to a form with spectral properties resembling the monomer absorbing at 670 nm but still in an aggregated state. This form of bacteriochlorophyll c showed no circular dichroism signal.     

Single crystal EPR study of VO(II)-doped magnesium potassium Tutton’s salt — Part 4

Single crystal EPR studies of VO(II)-doped magnesium potassium Tutton’s salt have been carried out at room temperature. The results indicate that the paramagnetic impurity has entered the lattice, both substitutionally and interstitially and the maximum hyperfine for the substitutional site along the a axis corresponds to the minimum hyperfine for interstitial site and vice versa. The spin Hamiltonian parameters obtained from single crystal data for these sites are: Site 1, g_||=1.954(1); g=1.998(1), A_||=19.80(2) mT; A=7.61(2) mT; Site 2, g_||=1.997(1); g=1.952(1), A_||=7.66(2) mT; A=19.85(2) mT. Superhyperfine from ligand protons have been observed at certain orientations for Site 2 impurity. Powder spectrum shows a set of eight parallel and perpendicular features indicating the presence of only one site and these values matched with Site 1 values. From these observations, it has been concluded that the two vanadyl impurities are approximately at right angles to each other. Cooling the sample to 77 K does not change the spectra appreciably. The admixture coefficients have been calculated from Site 1 data, which agree well with the reported values.     

STUDIES OF CHLOROPHYLL-CHLOROPHYLL AND CHLOROPHYLL-LIGAND INTERACTIONS BY VISIBLE ABSORPTION AND INFRARED SPECTROSCOPY AT LOW TEMPERATURES

Abstract— A comparison of the visible absorption and infrared spectra of various chlorophyll-chlorophyll (Chl) and Chi-nucleophile aggregates at room temperature and at low temperatures has been made. The IR data provide structural information indispensable for the interpretation of the visible spectra. As a necessary preliminary, it is shown that Chl a solutions in nonpolar solvents can be prepared by appropriate drying techniques that contain at a conservative estimate ≤ 3 mol % of water (i.e. Chl a/H₂O > 30:1). Very dry solutions of Chl a or Pyrochl a(≥ 10 mM) in toluene or methylcyclohexane-isopentane solution show only slight changes in visible spectra on cooling to 77 K. From IR, additional Chl-Chl aggregation occurs on cooling in methylcyclohexane-isopentane but not to a significant extent in toluene. Dilute (10 μM) solutions of Chl a or Pyrochl a in nonpolar solvents form a new absorption peak near 700 nm at low temperatures, which we attribute to traces of water in the solvent or other residual nucleophiles not removed during the Chl purification. Addition of stoichiometric amounts of water increases the size of the ?700 nm peak even in dilute Chl solutions. Chlorophyll a, Pyrochl a, but not pheophytin a are shown to interact with nucleophiles of the general type RXH (where R= H or alkyl, and X = O, N, or S). Such nucleophiles can coordinate to the Mg atom of one Chl molecule by lone pairs on O, N, or S, and hydrogen bond to oxygen donor functions in another Chl molecule. A ?0.1 M solution of Chl a or Pyrochl a in toluene containing 1.5 equivalents of ethanol is converted almost entirely to a species absorbing at ?700 nm at 77 K. Infrared spectroscopy shows conclusively that it is the keto C=O function that is involved in the cross-linking by hydrogen bonding, a conclusion supported by the observation that Pyrochl a forms a very similar red-shifted species at low temperatures, despite the absence of a carbomethoxy C=O function. n-Butylamine and ethanethiol interact in much the same way as does ethanol to form species red shifted to ?700 nm. A variety of possible structures for the low temperature forms is discussed, and the use of these red shifted species as paradigms for photoreaction center Chl is described.     

Electro-reflectance spectra of one-dimensional excitons in polydiacetylene crystals

Electro-reflectance spectra are reported for two spectroscopically-distinct forms of a polydiacetylene, poly(5,7-decadiyne-1,12-diol-bis phenylurethane). An electric field parallel to the polymer chain axis produces in both types of single crystals (1) a Stark shift of the main absorption peak and (2) a new absorption peak on the high energy side of the main peak. Using a one-dimensional exciton model, the main peak is assigned to the lowest ^tB_u exciton and the field-induced peak is assigned to the higher dipole-forbidden ^tA_g exciton, both associated with the one-dimensional π-electron bands on the polymer chains.     

CHLOROPHYLL-PROTEINS AND REACTION CENTER PREPARATIONS FROM PHOTOSYNTHETIC BACTERIA,ALGAE AND HIGHER PLANTS*,‡

Abstract— The use of sodium dodecyl sulfate to dissociate photosynthetic membranes followed by standard fractionation techniques yields chlorophyll-proteins and reaction center complexes with molecular weights of 500,000 or less. Much about the structure and function of photo-synthetic units in vivo can be deduced from the properties of the isolated complexes. The Bchl-protein from green bacteria is approximated by an incompletely filled sphere ? 80 Â in diameter consisting of four identical subunits. The five Bchl molecules in each subunit are 14 to 20Â apart. The related Chl a-proteins from a blue-green alga and various eukaryotic plants may have similar structural characteristics. The Chl a-protein from a blue-green alga contains one molecule of P700 per 60–90 Chl a molecules. The quantum requirement for P700 oxidation is 2.6 or less. The midpoint potential in various preparations ranges from 0.38 V to 0.42 V. Green algae and higher plants yield a Chl a-protein similar to that from the blue-green alga; in addition they yield another Chl-protein (mol. wt. = 2–3×10⁴) which contains an equal amount of Chl a and Chl b. These two Chl-proteins account for most of the chlorophyll in these organisms. Two photosynthetic bacteria (Rhodopseudomonas viridis and Chromatium) yield protein complexes containing Bchl, carotenoid, and bound cytochromes. The reaction center complex from R. viridis contains P960 (E_m, ₈= 0.39 to 0.42 V), cytochrome 558 (E_m,8= 0.33 V) and cytochrome 553 (E_m,7=— 0.02 V). Quantum requirements for P960 and C558 oxidation are ?2.2 and 3.0, respectively. Complex A from Chromatium contains Bchl 890, P883, cytochrome 556 (E_m,8= 0.34 V) and cytochrome 552 (E_m,7=?0.04 V). The quantum requirement for C556 oxidation is about 15. Both high- and low-potential cytochromes can donate electrons to the reaction center chlorophyll present in either complex. This fact supports the idea that only one kind of photochemical reaction center functions in photosynthetic bacteria. An hypothesis about the nature of the photosynthetic unit in purple bacteria is outlined.     

PROPERTIES OF ZINC AND MAGNESIUM METHYL BACTERIOPHEOPHORBIDE d AND THEIR AGGREGATES

Abstract— Light-harvesting bacteriochlorophylls are believed to he aggregated in oligomcric forms in chlorosomes of green photosynthetic bacteria. Zn and Mg methyl bacteriopheophorhides d (MBPd) were synthesized from chlorophyll a and studied as model compounds for bacteriochlorophyll d . Monomeric Zn and Mg MBPd in methanol have Q y absorption maxima at 650 nm and 657 nm and fluorescence decay lifetimes of 5.1 ns and 5.4 ns, respectively, comparcd to 5.6 ns for bactcriochlorophyll d . Zn and Mg MBPd both form oligomers in nonpolar solvents and exhibit Q, absorption maxima at 728 nm and 731 nm and fluorescence decay lifetimes of 14 ps and 19 ps, respectively, compared to 730 nm and 9 ps for similar bacteriochlorophyll d aggregates. One of the diastereomers at the 3^l position, R-Mg MBPd, forms intermediate-sized aggregated species that are equivalent to the dimer and a highly fluorescent species formed by bacteriochlorophylls c and d . The similarities of quantitative propcrtics between the model compounds, and the antenna pigments bacteriochlorophyll c and d indicate that Mg and Zn MBPd are good models for studying pigment interactions in chlorosomes and that the long hydrocarbon tail in the natural pigment is not required for oligomer formation. The dimer and the highly fluorescent species do not appear to be the building blocks of the oligomcr.     

1,15‐Penta­decane­diol

In the title compound, C₁₅H₃₂O₂, one of the terminal hydroxyl groups has a gauche conformation with respect to the hydro­carbon skeleton, while the other is trans. The mol­ecules lie parallel to the longest axis and form layers similar to those of the smectic A structure of liquid crystals. These features are similar to those of the homologues with an odd number of C atoms, but different from those with an even number.     

Studies on biaxial stretching of polypropylene film. IV. Overall orientation during one-step biaxial stretching and its application to the estimation of the principal refractive indices of isotactic polypropylene

Polypropylene film was biaxially stretched in one step in air at 140°C or 152°C, and the deformation was studied optically. A linear relation held between Δn_ss and v_A^?½ for v_A > 10, at both temperatures, where Δn_ss is the birefringence with respect to the normal to the film and v_A is the degree of stretching expressed as the factor by which the area of the film is increased. Extrapolation of data in this linear region yielded a value of 20 × 10^?3 for ?Δn_ss at infinite v_A. Since it is presumed that the polypropylene molecules lie completely parallel to the film surface when the film is stretched infinitely, ?Δn_ss at v_A^?½ = 0 must be just half Δn°, the intrinsic birefringence in the case of completely parallel orientation. Thus, Δn° must be 40 × 10^?3. This value was obtained experimentally in uniaxial stretching when the birefringence with respect to the direction of drawing was extrapolated to infinite extension. Similar relations held between n_p, the average of the refractive indices in the two stretching directions, and v_A, and between n_ss, the index normal to the film, and v_A. By similar extrapolations, (1/2)(n′_γ + n′_β) and n′_β = n*_α′ were estimated, and thence nα′ was obtained. Here, n′_α and n′_β are the refractive indices along the c axis (molecular chain axis) and b axis. All these optical parameters refer to a density of 0.900 g/cm³. Hence by applying a density correction to those values, the principal refractive indices and the intrinsic birefringence of polypropylene crystal were evaluated as follows: n_α = 1.5522, n_β = n*_α = 1.5106 and Δn_c° = 4.16 × 10^?3, where n*_α is the refractive index prependicular to the b and c axes of the crystal.     

Synthesis,Characterization and Crystal Structure of （2,2′—Bipyridine）（μ—maleato）copper（Ⅱ）Dihydrate

The title polymeric complex [Cu(C₄H₂O₄)(C₁₀H₈N₂)]_n·2nH₂O was prepared and its crystal structure was determined by X‐ray diffraction methods. The crystal belongs to space group P2₁/c with cell dimensions of a = 1.0104(1), b = 1.9952(2), c = 0.7357(2) nm, β= 98.38(2)° and Z = 4. The complex forms zig‐zag chains along crystallographic axis c via Cu—O (carboxyl) bond in the apical direction. Each repeated unit consists of a square pyramidal Cu(II) centre with one maleate dianion and one 2,2′‐bipyridine forming a basal plane. Adjacent chains link to each other by H‐bonding between carboxyl groups and crystalline water. The distance of 0.3482 nm between parallel bipyridine rings shows a π‐π stacking interaction. The title complex was also characterized by IR, UV and ESR spectra.     

Investigation in the Series of Substituted Butan- and Butenolides: XV. Transformations of 4-Hydroxy-2-butenolide in Water Media at Various pH Values

The 4-hydroxy-2-butenolide was established by polarographic method to undergo in water solutions fast tautomeric and acid-base transformations. Depending on pH of the medium the compound is present either as a cyclic irreducible form (at pH 0-4) or in the open-chain (carbonyl-containing) reducible forms (at pH > 4 and < 0). It is presumed that in going from the basic to strongly acidic medium and backwards occur consecutive equilibrium transitions of four species: cis--formylacrylic acid anion (A^-), its neutral molecule (HA) existing as linear and cyclic tautomers, and protonated forms (H₂A⁺ and H₃A⁺). The formation mechanism thereof is considered.