[Estimation of the Index Value of Dielectric Permeability inside the Membranes of Purple Bacteria].

The joint application of the precise X-ray data for isolated bacteriochlorophyll complexes of reaction centers and the fundamental formulae for the energy of interaction between two equal dipoles enabled us to suggest a new methodical approach for determination of the values of the index of dielectric permeability in the micro volume enclosing special pairs in Rhodobacter sphaeroides reaction centers. The most probable value for this parameter was thus determined within 1.66-1.76. This approach was generalized for the inner layer of the membranes of purple bacteria and yielded the index value about 1.70-1.85. It is argued that this range of dielectric permeability is adequate for bacterial and plant membranes as well. Low magnitude of this parameter contributes to higher efficiency of energy migration from vast light-harvesting chlorophyll "antenna" to the energy converting reaction centers and hence to higher efficiency of the whole photosynthesis.

Calculation of chromophore excited state energy shifts in response to molecular dynamics of pigment-protein complexes.

The absorption and energy transfer properties of photosynthetic pigments are strongly influenced by their local environment or "site." Local electrostatic fields vary in time with protein and chromophore molecular movement and thus transiently influence the excited state transition properties of individual chromophores. Site-specific information is experimentally inaccessible in many light-harvesting pigment-proteins due to multiple chromophores with overlapping spectra. Full quantum mechanical calculations of each chromophores excited state properties are too computationally demanding to efficiently calculate the changing excitation energies along a molecular dynamics trajectory in a pigment-protein complex. A simplified calculation of electrostatic interactions with each chromophores ground to excited state transition, the so-called charge density coupling (CDC) for site energy, CDC, has previously been developed to address this problem. We compared CDC to more rigorous quantum chemical calculations to determine its accuracy in computing excited state energy shifts and their fluctuations within a molecular dynamics simulation of the bacteriochlorophyll containing light-harvesting Fenna-Mathews-Olson (FMO) protein. In most cases CDC calculations differed from quantum mechanical (QM) calculations in predicting both excited state energy and its fluctuations. The discrepancies arose from the inability of CDC to account for the differing effects of charge on ground and excited state electron orbitals. Results of our study show that QM calculations are indispensible for site energy computations and the quantification of contributions from different parts of the system to the overall site energy shift. We suggest an extension of QM/MM methodology of site energy shift calculations capable of accounting for long-range electrostatic potential contributions from the whole system, including solvent and ions.

Ultrafast time-resolved spectroscopy of the light-harvesting complex 2 (LH2) from the photosynthetic bacterium Thermochromatium tepidum.

The light-harvesting complex 2 from the thermophilic purple bacterium Thermochromatium tepidum was purified and studied by steady-state absorption and fluorescence, sub-nanosecond-time-resolved fluorescence and femtosecond time-resolved transient absorption spectroscopy. The measurements were performed at room temperature and at 10 K. The combination of both ultrafast and steady-state optical spectroscopy methods at ambient and cryogenic temperatures allowed the detailed study of carotenoid (Car)-to-bacteriochlorophyll (BChl) as well BChl-to-BChl excitation energy transfer in the complex. The studies show that the dominant Cars rhodopin (N=11) and spirilloxanthin (N=13) do not play a significant role as supportive energy donors for BChl a. This is related with their photophysical properties regulated by long π-electron conjugation. On the other hand, such properties favor some of the Cars, particularly spirilloxanthin (N=13) to play the role of the direct quencher of the excited singlet state of BChl.

Structure of chlorosomes from the green filamentous bacterium Chloroflexus aurantiacus.

The green filamentous bacterium Chloroflexus aurantiacus employs chlorosomes as photosynthetic antennae. Chlorosomes contain bacteriochlorophyll aggregates and are attached to the inner side of a plasma membrane via a protein baseplate. The structure of chlorosomes from C. aurantiacus was investigated by using a combination of cryo-electron microscopy and X-ray diffraction and compared with that of Chlorobi species. Cryo-electron tomography revealed thin chlorosomes for which a distinct crystalline baseplate lattice was visualized in high-resolution projections. The baseplate is present only on one side of the chlorosome, and the lattice dimensions suggest that a dimer of the CsmA protein is the building block. The bacteriochlorophyll aggregates inside the chlorosome are arranged in lamellae, but the spacing is much greater than that in Chlorobi species. A comparison of chlorosomes from different species suggested that the lamellar spacing is proportional to the chain length of the esterifying alcohols. C. aurantiacus chlorosomes accumulate larger quantities of carotenoids under high-light conditions, presumably to provide photoprotection. The wider lamellae allow accommodation of the additional carotenoids and lead to increased disorder within the lamellae.

Dynamics of electron transfer from high-potential cytochrome c to bacteriochlorophyll dimer in photosynthetic reaction centers as probed using laser-induced temperature jump.

Laser-induced temperature jump experiments were used for testing the rates of thermoinduced conformational transitions of reaction center (RC) complexes in chromatophores of Chromatium minutissimum. The thermoinduced transition of the macromolecular RC complex to a state providing effective electron transport from the multiheme cytochrome c to the photoactive bacteriochlorophyll dimer within the temperature range 220-280 K accounts for tens of seconds with activation energy 0.166 eV/molecule. The rate of the thermoinduced transition in the cytochrome-RC complex was found to be three orders of magnitude slower than the rate of similar thermoinduced transition of the electron transfer reaction from the primary to secondary quinone acceptors studied in the preceding work (Chamorovsky et al. in Eur Biophys J 32:537-543, 2003). Parameters of thermoinduced activation of the electron transfer from the multiheme cytochrome c to the photoactive bacteriochlorophyll dimer are discussed in terms of cytochrome c docking onto the RC.

The architecture and function of the light-harvesting apparatus of purple bacteria: from single molecules to in vivo membranes.

This review describes the structures of the two major integral membrane pigment complexes, the RC-LH1 'core' and LH2 complexes, which together make up the light-harvesting system present in typical purple photosynthetic bacteria. The antenna complexes serve to absorb incident solar radiation and to transfer it to the reaction centres, where it is used to 'power' the photosynthetic redox reaction and ultimately leads to the synthesis of ATP. Our current understanding of the biosynthesis and assembly of the LH and RC complexes is described, with special emphasis on the roles of the newly described bacteriophytochromes. Using both the structural information and that obtained from a wide variety of biophysical techniques, the details of each of the different energy-transfer reactions that occur, between the absorption of a photon and the charge separation in the RC, are described. Special emphasis is given to show how the use of single-molecule spectroscopy has provided a more detailed understanding of the molecular mechanisms involved in the energy-transfer processes. We have tried, with the help of an Appendix, to make the details of the quantum mechanics that are required to appreciate these molecular mechanisms, accessible to mathematically illiterate biologists. The elegance of the purple bacterial light-harvesting system lies in the way in which it has cleverly exploited quantum mechanics.

Vibrational coherence in bacterial reaction centers with genetically modified B-branch pigment composition.

Femtosecond absorption difference spectroscopy was applied to study the time and spectral evolution of low-temperature (90 K) absorbance changes in isolated reaction centers (RCs) of the HM182L mutant of Rhodobacter (Rb.) sphaeroides. In this mutant, the composition of the B-branch RC cofactors is modified with respect to that of wild-type RCs by replacing the photochemically inactive BB accessory bacteriochlorophyll (BChl) by a photoreducible bacteriopheophytin molecule (referred to as PhiB). We have examined vibrational coherence within the first 400 fs after excitation of the primary electron donor P with 20-fs pulses at 870 nm by studying the kinetics of absorbance changes at 785 nm (PhiB absorption band), 940 nm (P*-stimulated emission), and 1020 nm (BA- absorption band). The results of the femtosecond measurements are compared with those recently reported for native Rb. sphaeroides R-26 RCs containing an intact BB BChl. At delay times longer than approximately 50 fs (maximum at 120 fs), the mutant RCs exhibit a pronounced BChl radical anion (BA-) absorption band at 1020 nm, which is similar to that observed for Rb. sphaeroides R-26 RCs and represents the formation of the intermediate charge-separated state P+ BA-. Femtosecond oscillations are revealed in the kinetics of the absorption development at 1020 nm and of decay of the P*-stimulated emission at 940 nm, with the oscillatory components of both kinetics displaying a generally synchronous behavior. These data are interpreted in terms of coupling of wave packet-like nuclear motions on the potential energy surface of the P* excited state to the primary electron-transfer reaction P*-->P+ BA- in the A-branch of the RC cofactors. At very early delay times (up to 80 fs), the mutant RCs exhibit a weak absorption decrease around 785 nm that is not observed for Rb. sphaeroides R-26 RCs and can be assigned to a transient bleaching of the Qy ground-state absorption band of the PhiB molecule. In the range of 740-795 nm, encompassing the Qy optical transitions of bacteriopheophytins HA, HB, and PhiB, the absorption difference spectra collected for mutant RCs at 30-50 fs resemble the difference spectrum of the P+ PhiB- charge-separated state previously detected for this mutant in the picosecond time domain (E. Katilius, Z. Katiliene, S. Lin, A.K.W. Taguchi, N.W. Woodbury, J. Phys. Chem., B 106 (2002) 1471-1475). The dynamics of bleaching at 785 nm has a non-monotonous character, showing a single peak with a maximum at 40 fs. Based on these observations, the 785-nm bleaching is speculated to reflect reduction of 1% of PhiB in the B-branch within about 40 fs, which is earlier by approximately 80 fs than the reduction process in the A-branch, both being possibly linked to nuclear wave packet motion in the P* state.

The role of carotenoids in the photoadaptation of the brown-colored sulfur bacterium Chlorobium phaeobacteroides.

The brown-colored sulfur bacterium Chlorobium (Cb.) phaeobacteroides 1549 (new name, Chlorobaculum limnaeum 1549) contains many kinds of carotenoids as well as bacteriochlorophyll (BChl) e. These carotenoids were identified with C18-high-performance liquid chromatography, absorption, mass and proton nuclear magnetic resonance spectroscopies and were divided into two groups: the first is carotenoid with one or two phi-end groups such as isorenieratene and beta-isorenieratene and the second is carotenoid with one or two beta-end groups such as p-zeacarotene, beta-carotene and 7,8-dihydro-beta-carotene. The latter 7,8-dihydro-beta-carotene was found to be a novel carotenoid in nature. OH-gamma-Carotene glucoside laurate and OH-chlorobactene glucoside laurate were also found as minor components. The distribution of BChl e homologs in Cb. phaeobacteroides cultivated under various light intensities did not change, but the carotenoid to BChl e ratio changed markedly: carotenoid with the phi-end group maintained the same ratio to BChl e, whereas that with the beta-end group increased with increasing light intensity. The cells cultured under low-light intensity contained more phi-end carotenoids than beta-end. In Cb. phaeobacteroides the wavelength of the Qy band of BChl e aggregates did not change. We suggested that Cb. phaeobacteroides photoadapts to light intensity by changing the carotenoid composition.

A protein conformational change associated with the photoreduction of the primary and secondary quinones in the bacterial reaction center.

A comparison is made between the PQA----P+QA- and PQAQB----P+QAQB-transitions in Rps. viridis and Rb. sphaeroides reaction centers (RCs) by the use of light-induced Fourier transform infrared (FTIR) difference spectroscopy. In Rb. sphaeroides RCs, we identify a signal at 1650 cm-1 which is present in the P+QA-minus-PQA spectrum and not in the P+QAQB(-)-minus-PQAQB spectrum. In contrast, this signal is present in both P+QA(-)-minus-PQA- and P+QAQB(-)-minus-PQAQB spectra of Rps. viridis RCs. These data are interpreted in terms of a conformational change of the protein backbone near QA (possible at the peptide C = O of a conserved alanine residue in the QA pocket) and of the different bonding interactions of QB with the protein in the RC of the two species.

Electrostatic control of charge separation in bacterial photosynthesis.

Electrostatic interaction energies of the electron carriers with their surroundings in a photosynthetic bacterial reaction center are calculated. The calculations are based on the detailed crystal structure of reaction centers from Rhodopseu-domonas viridis, and use an iterative, self-consistent procedure to evaluate the effects of induced dipoles in the protein and the surrounding membrane. To obtain the free energies of radical-pair states, the calculated electrostatic interaction energies are combined with the experimentally measured midpoint redox potentials of the electron carriers and of bacteriochlorophyll (BChl) and bacteriopheophytin (BPh) in vitro. The P+HL- radical-pair, in which an electron has moved from the primary electron donor (P) to a BPh on the 'L' side of the reaction center (HL), is found to lie approx. 2.0 kcal/mol below the lowest excited singlet state (P*), when the radical-pair is formed in the static crystallographic structure. The reorganization energy for the subsequent relaxation of P+HL- is calculated to be 5.0 kcal/mol, so that the relaxed radical-pair lies about 7 kcal/mol below P*. The unrelaxed P+BL- radical-pair, in which the electron acceptor is the accessory BChl located between P and HL, appears to be essentially isoenergetic with P*.P+BM-, in which an electron moves to the BChl on the 'M' side, is calculated to lie about 5.5 kcal/mol above P*. These results have an estimated error range of +/- 2.5 kcal/mol. They are shown to be relatively insensitive to various details of the model, including the charge distribution in P+, the atomic charges used for the amino acid residues, the boundaries of the structural region that is considered microscopically and the treatments of the histidyl ligands of P and of potentially ionizable amino acids. The calculated free energies are consistent with rapid electron transfer from P* to HL by way of BL, and with a much slower electron transfer to the pigments on the M side. Tyrosine M208 appears to play a particularly important role in lowering the energy of P+BL-. Electrostatic interactions with the protein favor localization of the positive charge of P+ on PM, one of the two BChl molecules that make up the electron donor.

In photosynthetic reaction centers, the free energy difference for electron transfer between quinones bound at the primary and secondary quinone-binding sites governs the observed secondary site specificity.

The secondary quinone-binding site (QB site) of bacterial reaction centers from Rhodobacter sphaeroides is generally regarded to be highly specific for its native ubiquinone-10 molecule. We demonstrate here that this is a misconception rooted in the kinetic methods used to assay for occupancy of a quinone in the QB site. We show that observance of occupancy of the QB site, revealed by kinetic assay, is sensitive to the free-energy difference for electron transfer between the quinone at the primary quinone-binding site (QA site) and the QB site (-delta G0e-). For many of the compounds previously tested for binding at the QB site, the -delta G0e- between QA and QB is too small to permit detection of the functional quinone in the QB site. With an increased -delta G0e- achieved by replacing the native ubiquinone-10 at the QA site with lower-potential quinones or by testing higher-potential QB candidates, it is shown that the QB site binds and functions with the unsubstituted 1,4-benzoquinone, 1,4-naphthoquinone, and 9,10-phenanthraquinone, as well as with their various substituted forms. Moreover, quinones with the ortho-carbonyl configuration appear to function in a similar manner to quinones with the para-carbonyl configuration.

Physiological and structural analysis of light-harvesting mutants of Rhodobacter sphaeroides.

Two mutants of Rhodobacter sphaeroides defective in formation of light-harvesting spectral complexes were examined in detail. Mutant RS103 lacked the B875 spectral complex despite the fact that substantial levels of the B875-alpha polypeptide (and presumably the beta polypeptide) were present. The B800-850 spectral complex was derepressed in RS103, even at high light intensities, and the growth rate was near normal at high light intensity but decreased relative to the wild type as the light intensity used for growth decreased. Mutant RS104 lacked colored carotenoids and the B800-850 spectral complex, as well as the cognate apoproteins. This strain grew normally at high light intensity and, as with RS103, the growth rate decreased as the light intensity used for growth decreased. At very low light intensities, however, RS104 would grow, whereas RS103 would not. Structural analysis of these mutants as well as others revealed that the morphology of the intracytoplasmic membrane invaginations is associated with the presence or absence of the B800-850 complex as well as of carotenoids. A low-molecular-weight intracytoplasmic membrane polypeptide, which may play a role in B800-850 complex formation, is described, as is a 62,000-dalton polypeptide whose abundance is directly related to light intensity as well as the absence of either of the light-harvesting spectral complexes. These data, obtained from studies of mutant strains and the wild type, are discussed in light of photosynthetic membrane formation and the abundance of spectral complexes per unit area of membrane. Finally, a method for the bulk preparation of the B875 complex from wild-type strain 2.4.1 is reported.

Optical properties and structure of tetrapyrroles. A report of a symposium held at the University of Konstanz, FRG, August 12-17, 1984.

Many basic life processes such as oxygen transport, electron transport, photosynthesis and plant development, are mediated by tetrapyrroles. It has long been recognized that optical and other physicochemical properties of tetrapyrroles are highly important not only for characterization of the various relevant systems but also to provide a key role in the understanding of their structural and functional aspects. The symposium described below was devoted mainly to recent advances in the optical and molecular properties of biologically important systems, involving both cyclic and open-chain tetrapyrroles. The topics presented and discussed ranged from physics and chemistry to plant biology and medicine. This interdisciplinary character of the meeting conforms with previous symposia also held in Konstanz: Protein-Ligand Interactions (1974) and Transport by Proteins (1978) [see FEBS Lett. (1975) 54, 1-4, and FEBS Lett. (1979) 103, 1-4]. In order to stimulate exchange of ideas, new experimental approaches and techniques, emphasis was placed on the discussions following each presentation, reports of which are also included in the book published. Preprints of the papers presented were distributed some time prior to the symposium. The symposium was mainly supported by the Stiftung Volkswagenwerk and the University of Konstanz. The meeting was based on 29 invited lectures which were divided into four sections.

The orientation of the primary donor in bacterial photosynthesis.

The triplet state EPR spectra of magnetically aligned whole cells of Rhodopseudomonas viridis and Rhodopseudomonas palustris display a marked dependence on the orientation of the static EPR field with respect to the alignment field direction. This observation implies that the primary donor species on which the triplets are localized are ordered within the membranes. We have developed a theoretical model for the system to enable calculation of the orientation of the magnetic axes of the primary donor species with respect to the membranes in which they reside. The triplet state spectra are generated by an ensemble of partially ordered magnetic systems and a computer simulation of the experimental results. The triplet orientation is very similar for the two organisms studied, where one axis lies predominantly in the plane of the membrane and the other two axes have approximately equal projections onto the normal to the membrane.