High-order fluorescence and exciton interaction in photosynthetic bacteria

We have observed fluorescence at visible wavelengths from chromatophores of photosynthetic bacteria excited with infrared radiation which we attribute to bacteriochlorophyll of the antenna system. The fluorescence is prompt (no delay greater than 5 ns). Its spectrum shows peaks at 445, 530 (broad) and 600 nm when excited with either 694 or 868 nm. Quantum yield is of the order of 10^?9. The dependence on intensity indicates generation by mainly third-order processes which could involve triplet states in combination with excited singlets. Second-order single-singlet fusion could also contribute. The high-order fluorescence can also be explained as arising from absorption of a second photon by singlet excited states.     

Tubular exciton models for BChl c antennae in chlorosomes from green photosynthetic bacteria

Exciton calculations on tubular pigment aggregates similar to recently proposed models for BChl c/d/e antennae in light-harvesting chlorosomes from green photosynthetic bacteria yield electronic absorption spectra that are super-impositions of linear J-aggregate spectra. While the electronic spectroscopy of such antennae differs considerably from that of linear J-aggregates, tubular exciton models (which may be viewed as cross-coupled J-aggregates) may be constructed to yield spectra that resemble that of the BChl c antenna in the green bacterium Chloroflexus aurantiacus. Highly symmetric tubular models yield absorption spectra with dipole strength distributions essentially identical to that of a J-aggregate; strong symmetry-breaking is needed to simulate the absorption spectrum of the BChl c antenna.Abbreviations BChl bacteriochlorophyll - [E,M] BChl c _S bacteriochlorophyll c with ethyl and methyl substituents in the 8- and 12-positions, and with stearol as the esterifying alcohol     

A master equation theory of fluorescence induction, photochemical yield, and singlet-triplet exciton quenching in photosynthetic systems.

A master equation theory is formulated to describe the dependence of the fluorescence yield (phi) in photosynthetic systems on the number of photons (Y) absorbed per photosynthetic unit (or domain). This theory is applied to the calculation of the dependence of the fluorescence yield on Y in (a) fluorescence induction, and (b) singlet exciton-triplet excited-state quenching experiments. In both cases, the fluorescence yield depends on the number of previously absorbed photons per domain, and thus evolves in a nonlinear manner with increasing Y. In case a, excitons transform the photosynthetic reaction centers from a quenching state to a nonquenching state, or a lower efficiency of quenching state; subsequently, absorbed photons have a higher probability of decaying by radiative pathways and phi increases as Y increases. In case b, ground-state carotenoid molecules are converted to long-lived triplet excited-state quenchers, and phi decreases as Y increases. It is shown that both types of processes are formally described by the same theoretical equations that relate phi to Y. The calculated phi (Y) curves depend on two parameters m and R, where m is the number of reaction centers (or ground-state carotenoid molecules that can be converted to triplets), and R is the ratio phi (Y leads to infinity)/(Y leads to 0). The finiteness of the photosynthetic units is thus taken into account. The m = 1 case corresponds to the "puddle" model, and m leads to infinity to the "lake," or matrix, model. It is shown that the experimental phi (Y) curves for both fluorescence induction and singlet-triplet exciton quenching experiments are better described by the m leads to infinity cases than the m = 1 case.     

Seven-fold exciton splitting of the 810-nm band in bacteriochlorophyll a-proteins from green photosynthetic bacteria

We report comparative absorbance and fourth derivative absorbance spectra of two different bacteriochlorophyll a-proteins at 5 K in each of two different cryogenic solvent mixtures. In previous studies at 5 K each protein was observed in only one of these mixtures (not the same one). For the protein from Prosthecochloris aestuarii strain 2K, whose structure is known, the solvent effect is relatively small; for the protein from Chlorobium limicola f. sp. thiosulfatophilum strain 6230 (Tassajara), the effect is much more pronounced. From these results together with earlier results at 300 K, we conclude there may be slight conformational differences of the Prosthecochloris protein between the crystalline form used for X-ray diffraction studies and that in a cryogenic solvent. By comparing spectral features of the two proteins in the same solvent, we are able for the first time to assign all seven of the expected exciton levels in each protein. These occur at 793, 801, 806, 810, 814, 819, and 825 nm in the Prosthecochloris protein, and at 793, 802, 806, 810, 816, 820, and 823 nm in the Chlorobium protein.     

Quinone-dependent delayed fluorescence from the reaction center of photosynthetic bacteria

Millisecond delayed fluorescence from the isolated reaction center of photosynthetic bacteria Rhodobacter sphaeroides was measured after single saturating flash excitation and was explained by thermal repopulation of the excited bacteriochlorophyll dimer from lower lying charge separated states. Three exponential components (fastest, fast, and slow) were found with lifetimes of 1.5, 102, and 865 ms and quantum yields of 6.4 x 10(-9), 2.2 x 10(-9), and 2.6 x 10(-9) (pH 8.0), respectively. While the two latter phases could be related to transient absorption changes, the fastest one could not. The fastest component, dominating when the primary quinone was prereduced, might be due to a small fraction of long-lived triplet states of the radical pair and/or the dimer. The fast phase observed in the absence of the secondary quinone, was sensitive to pH, temperature, and the chemical nature of the primary quinone. The standard free energy of the primary stable charge pair relative to that of the excited dimer was -910 +/- 20 meV at pH 8 and with native ubiquinone, and it showed characteristic changes upon pH and quinone replacement. The interaction energy ( approximately 50 meV) between the cluster of the protonatable groups around GluL212 and the primary semiquinone provides evidence for functional linkage between the two quinone binding pockets. An empirical relationship was found between the in situ free energy of the primary quinone and the rate of charge recombination, with practical importance in the estimation of the free energy levels from the easily available lifetime of the charge recombination. The ratio of the slow and fast components could be used to determine the pH dependence of the free energy level of the secondary stable charge pair relative to that of the excited dimer.     

Site inhomogeneity and exciton delocalization in the photosynthetic antenna

The influence of energy disorder on exciton states of molecular aggregates (the dimer and the circular aggregate) was analyzed. The dipole strength and inhomogeneous line shapes of exciton states were calculated by means of numerical diagonalization of Hamiltonian with diagonal energy disorder without intersite correlation. The disorder degree corresponding to destruction of coherent exciton states was estimated. The circular aggregates were treated as a model of light-harvesting antenna structures of photosynthetic bacteria. It was concluded that the site inhomogeneity typical for LH1 and LH2 complexes of purple bacteria cannot significantly influence the exciton delocalization over the whole antenna.Abbreviations BChl- bacteriochlorophyll - LH1 and LH2- core and peripheral light-harvesting complexes from purple bacteria - RC- reaction center     

Modifiable chromatophore proteins in photosynthetic bacteria.

The chromatophores of Chromatium vinosum, as well as six other photosynthetic bacteria, contained two or more proteins which were insoluble when heated in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (beta-ME). When the chromatophores were dissolved at room temperature in SDS-beta-ME, these proteins were present in the SDS-polyacrylamide gel electrophoresis profiles, but when the samples were dissolved at 100 degrees C, they were absent or considerably diminished. When one-dimensional gels of chromatophores solubilized at room temperature were soaked in the SDS-beta-ME solution and heated to 100 degrees C and the gels were run in a second dimension, the proteins became immobilized in the original first-dimension gel, where they could be detected by staining. The two major proteins so affected in C. vinosum had apparent molecular weights of 28,000 and 21,000. The chromatophores of several other photosynthetic bacteria also contained predominant proteins between 30,000 and 19,000 molecular weight, which became insoluble when heated in the presence of SDS and beta-ME. In at least two of the species examined, these appeared to be reaction center proteins. The conditions causing the proteins to become insoluble were complex and involved temperature, SDS concentration, and the presence of sulfhydryl reagents. The chromatophores of four of the Chromatiaceae species and two strains of one of the Rhodospirillaceae species examined had a protein-pigment complex that was visible in SDS-polyacrylamide gel profiles of samples dissolved at room temperature but was absent in samples dissolved at 100 degrees C.     

A picosecond pulse train study of exciton dynamics in photosynthetic membranes.

The fluorescence decay time of spinach chloroplasts at 77 degrees K was determined at 735 nm (corresponding to the photosystem I emission) using a train of 10-ps laser pulses spaced 10 ns apart. The fluorescence lifetime is constant at congruent to 1.5 ns for up to the fourth pulse, but then decreases with increasing pulse number within the pulse train. This quenching is attributed to triplet excited states, and it is concluded that triplet excitons exhibit a time lag of about 50 ns in diffusing from light harvesting antenna pigments to photosystem I pigments. The diffusion coefficient of triplet excitons is a least 300--400 times slower than the diffusion coefficient of singlet excitons in chloroplast membranes.     

A perturbed two-level model for exciton trapping in small photosynthetic systems.

The study of exciton trapping in photosynthetic systems provides significant information about migration kinetics within the light harvesting antenna (LHA) and the reaction center (RC). We discuss two random walk models for systems with weakly coupled pigments, with a focus on the application to small systems (10-40 pigments/RC). Details of the exciton transfer to and from the RC are taken into consideration, as well as migration within the LHA and quenching in the RC. The first model is obtained by adapting earlier local trap models for application to small systems. The exciton lifetime is approximated by the sum of three contributions related to migration in the LHA, trapping by the RC, and quenching within the RC. The second model is more suitable for small systems and regards the finite rate of migration within the LHA as a perturbation of the simplified model, where the LHA and the RC are each represented by a single pigment level. In this approximation, the exciton lifetime is the sum of a migration component and a single nonlinear expression for the trapping and quenching of the excitons. Numerical simulations demonstrate that both models provide accurate estimates of the exciton lifetime in the intermediate range of 20-50 sites/RC. In combination, they cover the entire range of very small to very large photosynthetic systems. Although initially intended for regular LHA lattices, the models can also be applied to less regular systems. This becomes essential as more details of the structure of these systems become available. Analysis with these models indicates that the excited state decay in LH1 is limited by the average rate at which excitons transfer to the RC from neighboring sites in the LHA. By comparing this to the average rate of transfer within the LHA, various structural models that have been proposed for the LH1 core antenna are discussed.     

Nitrogen fixation and hydrogen metabolism in photosynthetic bacteria.

The photosynthetic bacteria are found in a wide range of specialized aquatic environments. These bacteria represent important members of the microbial community since they are capable of carrying out two of the most important processes on earth, namely, photosynthesis and nitrogen fixation, at the expense of solar energy. Since the discovery that these bacteria could fix atmospheric nitrogen, there has been an intensification of studies relating to both the biochemistry and physiology of this process. The practical importance of this field is emphasized by a consideration of the tremendous energy input required for the production of artificial nitrogenous fertilizer. The present communication aims to briefly review the current state of knowledge relating to certain aspects of nitrogen fixation by the photosynthetic bacteria. The topics that will be discussed include a general survey of the nitrogenase system in the various photosynthetic bacteria, the regulation of both nitrogenase biosynthesis and activity, recent advances in the genetics of the nitrogen fixing system, and the hydrogen cycle in these bacteria. In addition, a brief discussion of some of some of the possible practical applications provided by the photosynthetic bacteria will be presented.     

Low-temperature fluorescence from single chlorosomes, photosynthetic antenna complexes of green filamentous and sulfur bacteria

Fluorescence spectra of single chlorosomes isolated from a green filamentous bacterium (Chloroflexus (Cfl.) aurantiacus) and a green sulfur bacterium (Chlorobium (Cb.) tepidum) were measured by using a confocal laser microscope at 13 K. Chlorosomes were frozen either in a liquid solution (floating chlorosome) or on a quartz plate after being adsorbed (adsorbed chlorosome). Fluorescence peak wavelengths were shorter for the adsorbed single chlorosomes than for the floating ones. Single floating Cfl. chlorosomes showed a distribution of fluorescence peak positions having a center at 759.0 nm with a full width at half maximum of 6.3 nm. Single floating Cb. chlorosomes showed a 782.7 nm center with a full width at half-maximum of 3.4 nm. The distribution shifted to the blue and became wider with increasing temperature, especially in Cb. chlorosomes, suggesting a large excitonic density of states just above the lowest level. Energy transfer from BChl-c aggregates to BChl-a molecules in the baseplate proteins was observed in the floating chlorosomes but not in the adsorbed ones. A positive correlation was found between the peak wavelength of BChl-c fluorescence and the intensity of BChl-a fluorescence in single Cfl. chlorosomes. The results suggest that the BChl-c aggregates with longer wavelengths of the fluorescence peaks have a more efficient F?rster-type energy transfer to the baseplate BChl-a.