RESONANCE RAMAN SPECTRA AND NORMAL MODE DESCRIPTIONS OF A BACTERIOCHLOROPHYLL a MODEL COMPLEX

Abstract— Resonance Raman (RR) spectra are reported for the bacteriochlorophyll a (BChl a) model complex, copper methylbacteriopheophorbide a (CuBPheo a). The spectra are acquired at a number of excitation wavelengths in the Q_y+ Q_x and B regions of the absorption spectrum. The RR data obtained for CuBPheo a are correlated to those previously reported for BChl a. The normal modes of the BChl structure are calculated by using the semiempirical quantum chemistry force field (QCFF/ PI) method of Warshel and Karplus [J. Am. Chem. Soc. 94 ,5612–5625 (1972)]. The vibrational data are used in conjunction with the calculations to obtain a cohesive assignment for the normal modes of BChl a with frequencies between 1100 and 1700 cm^-1. The forms of the normal modes of BChl a are predicted to be different both from those of chlorophyll a and from those of porphyrins.     

FOURIER TRANSFORM INFRARED SPECTROELECTROCHEMISTRY OF THE BACTERIOCHLOROPHYLL a ANION RADICAL

Abstract— Fourier transform infrared (FTIR) difference spectroscopy of the electrochemically generated anion radical of bacteriochlorophyll a was used to follow the molecular changes upon one-electron reduction. An IR spectroelectrochemical cell was constructed, allowing in situ electrolysis in connection with spectroscopic investigations from 200 to 10 000 nm. FTIR difference spectra of the BChl a anion formation in THF d₈ at U =+0.9 V (as determined by ferrocene calibration) were obtained. After complete formation of the radical, the reverse process was followed. Comparison of visible and IR spectra of the reduction and re-oxidation processes indicates that more than 90% of the BChl a anion could be formed with 90% of it being reversible. The main IR absorbance changes are observed for the conjugated and even for the non-conjugated C=O groups of BChl a . These results demonstrate the use of the combination of FTIR spectroscopy and electrochemistry for the characterization of radicals of the isolated pigments and of their in vivo bonding to the protein environment.     

EFFECTS OF SOLVENT ON THE FLUORESCENCE PROPERTIES OF BACTERIOCHLOROPHYLL a

Fluorescence lifetimes (τ_f) of bacteriochlorophyll a (BChl a ) have been measured by the method of time-correlated single-photon counting on dilute (1 μ M ) solutions of the pigment in 15 solvents. There is a pronounced dependence of τ_f on the nature of the solvent. Specifically, τ_f, is longer when the central magnesium is hexacoordinated than when pentacoordinated and shorter when the macrocycle is hydrogen-bonded than when it is not, but the latter effect is more pronounced. Both trends were confirmed by parallel studies on bacteriopheophytin a (BPheo a ). Because of the short lifetimes (˜ 2.2–3.6 ns), quenching of fluorescence by molecular oxygen is not a significant factor in aerated solutions of the bacterial pigments. However, reabsorption artifacts are non-negligible, which necessitates studies on dilute solutions. Fluorescence quantum yields (ø_f) have been estimated for BChl a in 13 solvents by comparing the observed fluorescence lifetimes with the radiative lifetimes calculated from the integrated absorption spectra.     

CONTROL OF SYNTHESIS OF REACTION CENTER BACTERIOCHLOROPHYLL IN PHOTOSYNTHETIC BACTERIA

Abstract— The ratio of total bacteriochlorophyll (BChl) to reaction center BChl has been determined in the wild-type and carotenoid-less mutants of Rhodopseudomonas spheroides and Rhodospirillum rubrum with various specific BChl contents. In wild-type R. spheroides the ratio increases as the specific BChl content increases; in the other strains the ratio is almost invariant.
The ability of the wild-type of R. spheroides to alter the size of the photosynthetic unit is apparently correlated with its multi-component BChl spectrum. The amount of reaction center BChl relative to the amount of the longest wavelength component of the spectrum is constant. The variation in the size of the photosynthetic unit is due to variation in the amount of this component relative to total BChl.
The size of the photosynthetic unit does not change as the specific BChl content decreases during growth under aerobic conditions of a culture grown previously under photosynthetic conditions.     

Physicochemical Studies of Demetalation of Light-harvesting Bacteriochlorophyll Isomers Purified from Green Sulfur Photosynthetic Bacteria

Demetalation kinetics of bacteriochlorophylls (BChls) c, d and e from green sulfur photosynthetic bacteria were studied under weakly acidic conditions. Demetalation rate constants of BChl e possessing a formyl group at the 7-position were significantly smaller than those of BChls c and d , which had a methyl group at this position. The activation energy of demetalation of 3¹ R -8,12-diethyl([E,E])-BChl e was 1.5-times larger than that of 3¹ R -[E,E]-BChl c . ¹⁵N-labeled 3¹ R -[E,E]-BChls c and e were purified from cells of green sulfur bacteria grown in a medium containing ¹⁵NH₄Cl, and their ¹⁵N NMR spectra were measured. The chemical shifts of N₂₁, N₂₂ and N₂₃ atoms of 3¹ R -[E,E]-BChl e were lower-field shifted than those of 3¹ R -[E,E]-BChl c , respectively, and especially the difference in chemical shifts of N₂₂ was significantly large. These results suggest that the electron-withdrawing formyl group at the 7-position of BChl e affected an electronic state of the chlorin macrocycle and caused BChl e to be more tolerant for removal of the central magnesium compared with BChls c and d .     

STUDIES ON THE INTERRELATIONSHIP AMONG THE INTENSITY OF A RAMAN MARKER BAND OF CAROTENOIDS, POLYENE CHAIN STRUCTURE, AND EFFICIENCY OF THE ENERGY TRANSFER FROM CAROTENOIDS TO BACTERIOCHLOROPHYLL IN PHOTOSYNTHETIC BACTERIA

Abstract Resonance Raman spectroscopy was used to study the polyene-chain structure of carotenoids in light-harvesting pigment-protein complexes from purple photosynthetic bacteria. When major carotenoids of Rhodobacter sphaeroides were incorporated into the light-harvesting complexes of Chromatium vinosum , they showed a relatively intense Raman band at 965 cm^-1 arising from the CH out-of-plane wagging modes of the polyene chain. This result was almost the same as that for the intrinsic carotenoids in Chromatium vinosum , but completely different from that for the intrinsic carotenoids in Rhodobacter sphaeroides . On the other hand, the intrinsic carotenoids of Chromatium vinosum lost the intensity of the 965 cm^-1 Raman band upon protein denaturation. These results support the view that the intensity of the 965 cm^-1 Raman band reflects distortion of the polyene chain, independent of its chemical structure. The polyene-chain distortion is affected by the apoprotein to which carotenoids are bound. The distortion of the polyene chain is correlated with a decrease in the efficiency of the energy transfer from carotenoids to bacteriochlorophyll.     

ENERGY TRANSFER BETWEEN THE PRIMARY DONOR BACTERIOCHLOROPHYLL AND CAROTENOIDS IN Rhodopseudomonas sphaeroides

Abstract— The temperature dependencies of the primary donor triplet state spectra are presented for the phorosynthetic bacteria Rhodopseudomonas sphaeroides wild type. GIC and R26. The data suggest that energy transfer from the primary donor triplet state to the reaction center carotenoid is dependent on the type of carotenoid present, reversible in the case of strain GIC, and best understood by a model depicting the kinetic processes that can occur between two potential energy surfaces; one representing the state ³BChl₂*Car and the other representing BChl₂³Car*. Furthermore, it is shown that the onset of spin lattice relaxation in the primary donor triplet is most likely coupled to the same energy vibrational mode as that which promotes triplet state energy transfer from the primary donor to the reaction center carotenoid     

INTERCONVERSION OF BACTERIOCHLOROPHYLL c AGGREGATES IN SOLID FILMS UPON ORGANIC VAPOR TREATMENT

Bacteriochlorophyll c (BChl c) solid films were prepared from a carbon tetrachloride solution on CaF₂ plates as artificial aggregates. Effects of organic vapor such as acetone and tetrahydrofuran (THF) on the BChl c films were studied by absorption and Fourier-transform infrared spectroscopy. Two major homologs (R[E,E]BChl c_F and R[P,E]BChl c_F) and one minor homolog (S[I,E]BChl c) isolated from the green photosynthetic bacterium Chlorobium limicola strain 6230 were examined for the experiments. The BChl c polymeric aggregates absorbing at739–753 nm similar to those in the chlorosome were induced for all homologs upon the treatment of BChl c solid film with acetone vapor. The 13¹-keto C=O stretching band in the R[E,E]BChl c_F solid film showed a downward shift from 1651 cm^?1to 1643 cm^?1 with a concomitant shift of the 3¹-OH stretching bands from 3337 and 3238 cm^?1 to 3163 cm^?1. It was suggested that the lower aggregates brought about by Mg…O=C(13¹) and (3¹)O…O=C(13¹) bonds were transformed into the higher aggregates strongly hydrogen-bonded in a Mg…(3¹)O-H…O=C(13^l) interaction. They were transformed to a monomer-like form absorbing at 667 nm upon exposure to THF vapor and were reversibly converted to the higher aggregates upon removal of THF molecules in vacuo.     

The Structure of the Aggregate Form of Bacteriochlorophyll c Showing the Qy Absorption above 740 nm as Determined by the Ring-current Effects on 1H and 13C Nuclei and by 1H–1H Intermolecular NOE Correlations

¹³C-enriched bacteriochlorophyll c (S[I, E] BChl c _F) was suspended in a 1:3 mixture of methylene chloride and carbon tetrachloride to form an aggregate showing the Q_y absorption above 740 nm; changes in the ¹³C chemical shifts were traced when methanol was titrated to dissolve the aggregate, and then, the changes were correlated to the ring-current effects due to the neighboring macrocycles in the aggregate. A pair of aggregate structures has been proposed based on the ring-current effects on both ¹H and ¹³C nuclei; the monomeric units are stacked together to form an inclined column with different sliding directions, in which the y-axis of the molecule is parallel to the long axis of the column. In order to confirm this pair of models, the ring-current effects on the ¹H and ¹³C nuclei were calculated based on both the magnetic-dipole and the loop-current approximations. Further, an application of three-dimensional F1 ¹³C-edited F3 ¹³C-filtered heteronuclear single-quantum correlation-nuclear Overhauser effect spectroscopy to the above aggregate consisting of a 1:1 mixture of ¹³C-labeled and unlabeled BChl c succeeded in detecting selectively the ¹H–¹H intermolecular nuclear Overhauser effect correlations, which established the coexistence of the above pair of stacked structures in the aggregate.     

Aggregation of the Light-Harvesting Complex in Intact Leaves of Tobacco Plants Stressed by CO2 Deficit

Pronounced aggregation of the photosystem II light-harvesting complex (LHC II) was observed in low-lightgrown tobacco plants stressed with a strong CO₂ deficit for 2–3 days. The LHC II aggregates showed a typical band at 697–700 nm (F₆₉₉) in low-temperature emission spectra. Its excitation spectrum corresponded to that of detergent-solubilized LHC II. Formation of F₆₉₉ in stressed plants was not reversed in the dark and leaves did not contain any zeaxanthin showing that neither a light-induced transthylakoid pH gradient nor zeaxanthin was required for LHC II aggregation. The CO₂-stressed plants showed clear signs of photodamage: depression of the potential yield of photosystem II photochemistry (F,/F_M) by 50–70% and a decline in chlorophyll content by 10–15%. Therefore, we propose that the photodamage to the photosynthetic apparatus is the cause of the LHC II aggregation in plants. The F₆₉₉ exhibited a reversible decrease of its intensity upon irradiation of leaves with intensive light. There was no or only slight decrease around 700 nm in unstressed plants. The nonphotochemical quenching of chlorophyll fluorescence showed the opposite relation, being higher before than after the strong CO₂ deficit. This discrepancy was likely related to the different LHC II aggregation state in control and stressed plants.     

PHOTOSENSITIZING PROPERTIES OF BACTERIOCHLOROPHYLLIN a and BACTERIOCHLORIN a, TWO DERIVATIVES OF BACTERIOCHLOROPHYLL a

Abstract The photosensitizing properties of two water soluble derivatives of bacteriochlorophyll a , bacteriochlorophyllin a and bacteriochlorin a (lacking the central Mg-ion) were investigated and compared to those of hematoporphyrin derivatives. At physiological pH the oxygen consumption rate of histidine, tryptophan, dithiothreitol and guanosine upon illumination was 3 to 4 times higher when bacteriochlorin a was used as photosensitizer than when hematoporphyrin derivatives were used. Especially bacteriochlorin a proved to be an effective sensitizer for the killing of L929 cells. Because bacteriochlorin a has an absorption maximum at 765 nm in phosphate buffered saline (allowing a light penetration in tissue about ten times larger than at 630 nm) and a high molar absorption coefficient (32 000 M cm⁻¹) it has promising possibilities for the application in photodynamic therapy.     

ENERGY CONVERSION IN THE DECAY OF TRIPLET LUMIFLAVIN IN THE PRESENCE OF FERRI- AND FERROCYANIDE

Abstract— Flash photolysis at 450 nm has been used to study the quenching of the excited triplet state of lumiflavin and the transient species formed in subsequent reactions in deaerated phosphate buffer (pH 6.9).
The effect of the presence of ferricyanide on the life time of triplet lumiflavin has been studied. The results suggest an energy transfer reaction without concurrent electron transfer reactions. The rate constant for the process was 2.8 times 10⁹ M ^-1 s^-1. The analogous reaction with ferrocyanide could not be observed because of the efficient electron transfer reaction (δG = -20.6 kcal mol^-1) leading to the formation of the semireduced lumiflavin and ferricyanide. The rate constant for this reaction was 3.3 times 10⁹ M ^-1 s^-1. The semireduced lumiflavin radical was found to disappear in a second order reaction with a rate constant of 1.7 times 10⁹ M ^-1 s^-1. It was found to react with ferricyanide with a rate constant of 0.7 times 10⁹ M ^-1 s^-1.
A model for the various photochemical and photophysical processes involved in the decay and quenching of the lumiflavin triplet state is suggested and discussed.     

Structure-Based Calculations of the Optical Spectra of the LH2 Bacteriochlorophyll-Protein Complex from Rhodopseudomonas acidophila

Abstract— The molecular structure of the light-harvesting complex 2 (LH2) bacteriochlorophyll-protein antenna complex from the purple non-sulfur photosynthetic bacterium Rhodopseudomonas acidophila , strain 10050 provides the positions and orientations of the 27 bacteriochlorophyll (BChl) molecules in the complex. Our structure-based model calculations of the distinctive optical properties (absorption, CD, polarization) of LH2 in the near-infrared region use a point-monopole approximation to represent the BChl Q_y transition moment. The results of the calculations support the assignment of the ring of 18 closely coupled BChl to B850 (BChl absorbing at 850 nm) and the larger diameter, parallel ring of 9 weakly coupled BChl to B800. All of the significantly allowed transitions in the near infrared are calculated to be perpendicular to the C9 symmetry axis, in agreement with polarization studies of this membrane-associated complex. To match the absorption maxima of the B800 and B850 components using a relative permittivity (dielectric constant) of 2.1, we assign different site energies (12 500 and 12260 cm⁻¹, respectively) for the Q_y transitions of the respective BChl in their protein binding sites. Excitonic coupling is particularly strong among the set of B850 chromophores, with pairwise interaction energies nearly 300 cm between nearest neighbors, comparable with the experimental absorption bandwidths at room temperature. These strong interactions, for the full set of 18 B850 chromophores, result in an excitonic manifold that is 1200 cm⁻¹ wide. Some of the upper excitonic states should result in weak absorption and perhaps stronger CD features. These predictions from the calculations await experimental verification.     

SPECTRA AND EXTINCTION COEFFICIENTS OF NEAR-INFRARED ABSORPTION BANDS IN MEMBRANES OF Rhodobacter sphaeroides MUTANTS LACKING LIGHT-HARVESTING AND REACTION CENTER COMPLEXES

Abstract— Membranes from Rhodobacter (formerly Rhodopseudomonas) sphaeroides mutant strains that lack one or more of the bacteriochlorophyll a (BChl)-protein complexes were used to obtain spectra and molar extinction coefficients of the near-IR absorption bands. The strains examined were NF57 which lacks the B875 light-harvesting and reaction center complexes, and M21 which lacks the B800–850light-harvesting complex. The extinction coefficients obtained for the B800, B850 and B875 bands were 226 ± 10, 170 ± 5, and 118 ± 5 m M ^-1cm^-1, respectively, in reasonable agreement with values reported for detergent-isolated complexes (Clayton, R. K. and B. J. Clayton, Proc. Natl. Acad. Sci. USA 78 ,5583–5587, 1981). The results also demonstrated that detergent solubilization altered the spectra, causing a band broadening on the blue side of the B875 and B800 peaks. The data obtained from the analysis of the mutant strains were used for deconvolution of the BChl species in membranes of the wild-type. A short BASIC computer program for performing this deconvolution is included.     

EFFECTS OF QUENCHING AGENTS ON THE PHOTODYNAMICALLY-INDUCED CHEMOTACTIC RESPONSE OF THE COLORLESS FLAGELLATE Polytomella magna

Abstract In the presence of the photosensitizer riboflavin at high fluence rates a photoproduct, most probably H₂O₂, is formed which causes negative phototaxis in the colorless flagellate Polytomella magna . The aim of this study was to find out whether H₂O₂ is produced in a type I or II reaction. As has been shown, ¹O₂ quenchers either do not influence the photodynamic action of riboflavin (furfuryl ethanol, DPBF, l -histidine, crocetin) or show slight quenching effects only at very high concentrations ≧ 10⁻² M (DABCO, DMF, imidazole). D₂O is toxic to P. magna even in 1:1 and 1:2 mixtures with H₂O. On the other hand, the quenching effect of 1,4-benzoquinone, highly indicative for the type I pathway, is more than two orders of magnitude stronger than the one of the above mentioned ¹O₂ quenchers. The results suggest that H₂O₂ is produced in a type I reaction. Superoxide does not seem to be involved since superoxide dismutase does not diminish the photodynamic effect of riboflavin.     

POLARIZED SPECTRA OF ORIENTED PIGMENT-LIPOPROTEIN COMPLEXES FROM THE PURPLE BACTERIUM: Chromatium minutissimum

Abstract— Polarized absorption, fluorescence and photoacoustic spectra of bacteriochlorophyll (BChl)-lipoprotein complexes from the purple bacterium Chromatium minutissimum oriented in stretched polyvinylalcohol films were measured at room temperature and 85 K. The preparations contain large amounts of the B800-820 antenna complexes. From polarized absorption spectra taken under various light beam incidence angles with respect to the film plane, conclusions concerning arrangement of pigment molecules in B800-820 complex are obtained. The transition moments of the BChl Q_y band are not exactly parallel to the membrane plane. It seems that there are pools of differently oriented BChl chromophores absorbing in both 800 nm and 820 nm regions. Change in temperature strongly influences linear dichroism of carotenoids and BChl Q_y bands. The reversible changes in absorption, linear dichroism and photoacoustic spectra caused by the variation in sample temperature suggest strongly the reversible twisting of carotenoid molecules, related probably to modification of the interactions between carotenoids and proteins. Various carotenoids exhibit different yield of thermal deactivation and this yield is also temperature dependent.     

CHEMILUMINESCENCE AND THE FORMATION OF SINGLET OXYGEN IN THE OXIDATION OF CERTAIN POLYPHENOLS AND QUINONES

Abstract— The possibility of ¹O₂ (¹Δ_g) participation in the oxidation of polyphenols and quinones has been investigated in two systems: (1) the system involving autooxidation leading to oxidative polymerization and destruction, and (2) the modified Trautz-Schorigin reaction, i.e. oxidation of polyphenols and HCHO with H₂O₂ in concentrated alkaline solutions. The red band with maximum at 635 nm observed in chemiluminescence of pyrocatechol, adrenaline, pyrogallol, gallic acid, adrenochrome and p -benzoquinone corresponds to the transition 2O₂(¹Δ_g) → 2O₂(³Σ^-_g). Emission bands in the range 475–540 nm arise from the superposition of the 2O₂(¹Δ_g) → 2O₂(³Σ^-_g) transition and radiative deactivation of excited oxidation products. In system (2) chemiluminescence has a broad band from 580 nm beyond 800 nm and much higher intensity than in system (1). Formaldehyde was found to enhance light emission in system (1) by a factor of about 30. The influence of solvents, including D₂O in which ¹O₂ has varying lifetimes, on kinetics of chemiluminescence as well as quenching effect of β-carotene, hydroquinone, cysteine, bilirubin and biliverdin strongly support the involvement of ¹O₂ in the chemiluminescence of both systems.     

INVESTIGATION OF THE ALLOMERIZATION REACTION OF CHLOROPHYLL a: USE OF DIODE ARRAY HPLC, MASS SPECTROMETRY AND CHEMOMETRIC FACTOR ANALYSIS FOR THE DETECTION OF EARLY PRODUCTS

Abstract –The products of chlorophyll allomerization in methanol were isolated and analyzed by open column sucrose chromatography, liquid chromatography mass spectrometry (LCMS) and DAD-HPLC (diode-array high-performance liquid chromatography). Four main bands were found with molecular ions of (a) 908, (b) 938, (c) 938 and (d) 938, consistent with the structures (a) 13²-hydroxy-chlorophyll a (II), (b) and (c) Mg(II)-3¹,3²-didehydro-15¹-hydroxy-15¹-methoxy-rhodochlorin-15 acetic acid δ-lactone 15²-methyl 17³-phytyl ester and its epimer (III) and (d) Mg(II)-3¹,3²-didehydro-rhodochlorin-15-glyoxylic acid 13¹,15²-dimethyl 17³-phytyl ester (IV), evidence enhanced by UV/visible spectroscopy, chromatographic coelutions and chemometrics. Chlorophyll a was degraded both in the dark and light, under O₂ and N². DAD-HPLC of the resultant degradation mixtures were analyzed using the chemometric heuristic-evolving latent projection method for resolution. Ultraviolet/visible spectra of II and III are reproducibly extracted from the mixtures after a short degradation time, whereas III and IV are the dominant compounds after longer degradation times. Changes in relative elution order of IV using open column chromatography and reverse-phase HPLC are established. A possible allomerization pathway is proposed.     

FURTHER IN VIVO STUDIES ON THE PARTICIPATION OF SINGLET OXYGEN IN THE PHOTODYNAMIC INACTIVATION AND INDUCTION OF GENETIC CHANGES IN SACCHAROMYCES CEREVISIAE

Abstract —In vivo participation of singlet excited oxygen (¹O₂, ¹Δ9) in the photodynamic inactivation and induction of genetic changes (gene conversion) in acridine orange-sensitized yeast cells was investigated by using N₃^-, an efficient ¹O₂ quencher, and D²O, a known agent for the enhancement of the lifetime of ¹O₂. The addition of N₃^- protected the cells from both photodynamic actions. From an analysis of the concentration-dependent protection, about 80% of the induction of the genetic change is explainable on the basis of ¹O₂ mechanism. The quantitative estimation of the N₃^- protection in the inactivation was not possible because of the sigmoidal nature of the inactivation curve. The replacement of H₂O with D₂O during illumination was effective in enhancing the photodynamic inactivation but almost completely ineffective for the gene conversion induction. The deuterium effect with the cell system was clearly not as large as would be expected from in vitro experiments. This, however, could be explained from the kinetic consideration that natural quenchers of ^lO₂ in the cell would mask the deuterium effect. By experiments with different cell stages it was demonstrated that these two modifying effects were dependent on the intracellular reaction environment. The conclusion is that ¹O₂ must be the major intermediate responsible for the photodynamic actions in acridine orangesensitized yeast cells.     

THE EFFECT OF CHEMICAL STRUCTURE ON THE PHOTOSENSITIZING EFFICIENCIES OF PORPHYRINS

Abstract— A kinetic investigation was performed on the photooxidation of methionine sensitized by various porphyrins at different oxygen concentrations. The rate of photooxidation was found to be strongly dependent on the nature of the sensitizer. In the case of hematoporphyrin, chelation of Mg²⁺ and Zn²⁺ and especially of Cu²⁺ and Fe²⁺ caused a significant decrease of the photosensitizing efficiency. Fluorescence and/or flash photolysis studies showed that such a decrease is ascribed to an enhancement of the non-radiative decay of the first excited singlet state as well as to a reduction of the triplet lifetime. The sensitizing efficiency is also dependent on the nature of the porphyrin side chains. A reaction mechanism involving ¹O₂ as the oxidizing agent is proposed.