Characterization of the light harvesting antennas of photosynthetic purple bacteria by Stark spectroscopy. 2. LH2 complexes: influence of the protein environment

We have performed low-temperature Stark spectroscopy on a variety of different LH2 complexes from four photosynthetic bacteria, with the aim of characterizing the electric field response of the B800 and B850 absorption properties as a function of the protein environment. The following LH2 complexes were investigated: B800-850 and B800-820 of Rhodopseudomonas (Rps) acidophila; B800-850, B800-840 (αTyr+13→Phe), and B800-826 (αTyr+13→Phe, αTyr+14→Leu) of Rhodobacter (Rb.) sphaeroides; B800-850 and B800-830 (obtained at high LDAO) of Ectothiorhodospira sp.; and B800-850 of Rhodospirillum (Rsp.) molischianum. For all these cases the spectral blue shift of B850 has been assigned to the loss hydrogen-bonding interaction with the acetyl carbonyl of bacteriochlorophyll a. |Δμ| values for the 850 nm bands as well as for the blue-shifted bands are all on the order of 3-4.5 D/f. The loss of hydrogen-bonding interactions has only small effects on |Δμ| in these complexes. The values of the difference polarizability, Tr(Δαa), are large (600-1400 Å3/f2). The results are discussed in terms of crystal-structure-based models for LH2, in which pigment-pigment and pigment-protein interactions are considered; strong pigment-pigment interactions were found to be especially important. The values of |Δμ| for the 800 nm band are small, 1.0-1.5 D/f for LH2 complexes from Rb. sphaeroides and Rps. acidophila. However, in Rsp. molischianum and Ectothiorhodospira sp. |Δμ| values are much larger, of the order of 3 D/f. The difference in the B800 band is assigned to the difference in orientation of the B800 pigments in Rsp. molischianum and Ectothiorhodospira sp., as compared to the Rps. acidophila and Rb. sphaeroides. Due to the difference in orientation, the interactions of the Bchl a with the surrounding protein and neighboring carotenoid pigments are also not identical.Peer Reviewe

In vivo reconstitution of a homodimeric cytochrome b559 like structure: The role of the N-terminus a-subunit from Synechocystis sp. PCC 6803

The cytochrome b559 is a heme-bridged heterodimeric protein with two subunits, a and ß. Both subunits from Synechocystis sp. PCC 6803 have previously been cloned and overexpressed in Escherichia coli and in vivo reconstitution experiments have been carried out. The formation of homodimers in the bacterial membrane with endogenous heme was only observed in the case of the ß-subunit (ß/. ß) but not with the full length a-subunit. In the present work, reconstitution of a homodimer (a/. a) cytochrome b559 like structure was possible using a chimeric N-terminus a-subunit truncated before the amino acid isoleucine 17, eliminating completely a short amphipathic a-helix that lays on the surface of the membrane. Overexpression and in vivo reconstitution in the bacteria was clearly demonstrated by the brownish color of the culture pellet and the use of a commercial monoclonal antibody against the fusion protein carrier, the maltoside binding protein, and polyclonal antibodies against a synthetic peptide of the a-subunit from Thermosynechococcus elongatus. Moreover, a simple partial purification after membrane solubilization with Triton X-100 confirmed that the overexpressed protein complex corresponded with the maltoside binding protein-chimeric a-subunit cytochrome b559 like structure. The features of the new structure were determined by UV-Vis, electron paramagnetic resonance and redox potentiometric techniques. Ribbon representations of all possible structures are also shown to better understand the mechanism of the cytochrome b559 maturation in the bacterial cytoplasmic membrane

Site Energies of Active and Inactive Pheophytins in the Reaction Center of Photosystem II from Chlamydomonas Reinhardtii

31 Pags. The definitive version is available at: http://pubs.acs.org/journal/jpcbfkIt is widely accepted that the primary electron acceptor in various Photosystem II (PSII) reaction centers (RCs) is pheophytin a (Pheo a) within the D1 protein (PheoD1), while PheoD2 (within the D2 protein) is photochemically inactive. The Pheo site energies, however, have remained elusive, due to inherent spectral congestion. While most researchers over the last two decades assigned the Qy-states of PheoD1 and PheoD2 bands near 678–684 nm and 668–672 nm, respectively, recent modeling [Raszewski et al. Biophys. J. 2005, 88, 986–998; Cox et al. J. Phys. Chem. B 2009, 113, 12364–12374] of the electronic structure of the PSII RC reversed the location of the active and inactive Pheos, suggesting that the mean site energy of PheoD1 is near 672 nm, whereas PheoD2 (~677.5 nm) and ChlD1 (~680 nm) have the lowest energies (i.e., the PheoD2-dominated exciton is the lowest excited state). In contrast, chemical pigment exchange experiments on isolated RCs suggested that both pheophytins have their Qy absorption maxima at 676–680 nm [Germano et al. Biochem. 2001, 40, 11472–11482; Germano et al. Biophys. J. 2004, 86, 1664–1672]. To provide more insight into the site energies of both PheoD1 and PheoD2 (including the corresponding Qx transitions, which are often claimed to be degenerate at 543 nm) and to attest that the above two assignments are most likely incorrect, we studied a large number of isolated RC preparations from spinach and wild-type Chlamydomonas reinhardtii (at different levels of intactness) as well as the Chlamydomonas reinhardtii mutant (D2-L209H), in which the active branch PheoD1 is genetically replaced with chlorophyll a (Chl a). We show that the Qx-/Qy-region site-energies of PheoD1 and PheoD2 are ~545/680 nm and ~541.5/670 nm, respectively, in good agreement with our previous assignment [Jankowiak et al. J. Phys. Chem. B 2002, 106, 8803–8814]. The latter values should be used to model excitonic structure and excitation energy transfer dynamics of the PSII RCs.Partial support to B.N. (involved in calculations) was provided by the NSF EPSCoR Grant. V.Z. (involved in writing the manuscript) acknowledges support by NSERC. R.T.S., R.P., and M.S. were involved in the design and preparation of D2-mutant and RCs. They acknowledge support from USDOE, Photosynthetic Antennae Research Center (R.T.S.), MICIN (Grant AGL2008-00377) in Spain (R.P.), and the U.S. Department of Energy’s Photosynthetic Systems Program within the Chemical Sciences, Geosciences, and Biosciences Division of the Office of Basic Energy Sciences under NREL Contract #DE-AC36-08-GO28308 (M.S.).Peer reviewe

Surface science of soft scorpionates

The chemisorption of the soft scorpionate Li[PhTmMe] onto silver and gold surfaces is reported. Surface enhanced Raman spectroscopy in combination with the Raman analysis of suitable structural models, namely, [Cu(κ3-S,S,S-PhTmMe)(PCy3)], [Ag(κ3-S,S,S-PhTmMe)(PCy3)], [Ag(κ2-S,S-PhTmMe)(PEt3)], and [Au(κ1-S-PhTmMe)(PCy3)], are employed to identify the manner in which this potentially tridentate ligand binds to these surfaces. On colloidal silver surface-enhanced Raman spectroscopy (SERS) spectra are consistent with PhTmMe binding in a didentate fashion to the surface, holding the aryl group in close proximity to the surface. In contrast, on gold colloid, we observe that the species prefers a monodentate coordination in which the aryl group is not in close proximity to the surface

Supramolecular arrangement of Rhodospirillum rubrum B880 holochrome as studied by radiation inactivation and electron paramagnetic resonance.

Oxidation of the B880 antenna holochrome gives rise to a 3.8-G linewidth electron paramagnetic resonance (EPR) signal that is considerably narrower than the 13-G signal of monomeric bacteriochlorophyll (Bchl) cation. Radiation inactivation was used to verify a model according to which this linewidth narrowing is due to delocalization over several Bchl molecules. Chromatophores of the photoreaction centerless mutant F24 of Rhodospirillum rubrum were subjected to different doses of gamma-radiation. This induced not only a decay of the EPR signal amplitude but also its linewidth broadening. According to target theory, the induced amplitude decay of the EPR signal had a target size of 10.5 kDa. This is attributed to an elementary structure (alpha1beta1Bchl2), whose number in the membrane would limit the rate of encounter with ferricyanide and thus the formation of unpaired spins. We applied Bernoulli statistics to predict, for a given survival probability of the signal, the number of surviving elementary structures in aggregates of (alpha1beta1Bchl2)n where n was varied from 4 to 7. Using an equation that predicted the Bchl special pair in the photo-reaction center, we were able to simulate the observed relationship between the EPR linewidth and the dose of radiation. The best fit was obtained with a hexameric structure alpha1beta1Bchl2)6

The GmFAD7 gene family from soybean : identification of novel genes and tissue-specific conformations of the FAD7 enzyme involved in desaturase activity

The FAD7 gene encodes a omega3 fatty acid desaturase which catalyses the production of trienoic fatty acids (TAs) in plant chloroplasts. A novel GmFAD7 gene (named GmFAD7-2) has been identified in soybean, with high homology to the previously annotated GmFAD7 gene. Genomic sequencing analysis together with searches at the soybean genome database further confirmed that both GmFAD7 genes were located in two different loci within the soybean genome, suggesting that the soybean omega3 plastidial desaturase FAD7 is encoded by two different paralogous genes. Both GmFAD7-1 and GmFAD7-2 genes were expressed in all soybean tissues examined, displaying their highest mRNA accumulation in leaves. This expression profile contrasted with GmFAD3A and GmFAD3B mRNA accumulation, which was very low in this tissue. These results suggested a concerted control of plastidial and reticular omega3 desaturase gene expression in soybean mature leaves. Analysis of GmFAD7 protein distribution in different soybean tissues showed that, in mature leaves, two bands were detected, coincident with the higher expression level of both GmFAD7 genes and the highest 18:3 fatty acid accumulation. By contrast, in seeds, where FAD7 activity is low, specific GmFAD7 protein conformations were observed. These GmFAD7 protein conformations were affected in vitro by changes in the redox conditions of thiol groups and iron availability. These results suggest the existence of tissue-specific post-translational regulatory mechanisms affecting the distribution and conformation of the FAD7 enzymes related with the control of its activity

Site Energies of Active and Inactive Pheophytins in the Reaction Center of Photosystem II from Chlamydomonas Reinhardtii

31 Pags. The definitive version is available at: http://pubs.acs.org/journal/jpcbfkIt is widely accepted that the primary electron acceptor in various Photosystem II (PSII) reaction centers (RCs) is pheophytin a (Pheo a) within the D1 protein (PheoD1), while PheoD2 (within the D2 protein) is photochemically inactive. The Pheo site energies, however, have remained elusive, due to inherent spectral congestion. While most researchers over the last two decades assigned the Qy-states of PheoD1 and PheoD2 bands near 678–684 nm and 668–672 nm, respectively, recent modeling [Raszewski et al. Biophys. J. 2005, 88, 986–998; Cox et al. J. Phys. Chem. B 2009, 113, 12364–12374] of the electronic structure of the PSII RC reversed the location of the active and inactive Pheos, suggesting that the mean site energy of PheoD1 is near 672 nm, whereas PheoD2 (~677.5 nm) and ChlD1 (~680 nm) have the lowest energies (i.e., the PheoD2-dominated exciton is the lowest excited state). In contrast, chemical pigment exchange experiments on isolated RCs suggested that both pheophytins have their Qy absorption maxima at 676–680 nm [Germano et al. Biochem. 2001, 40, 11472–11482; Germano et al. Biophys. J. 2004, 86, 1664–1672]. To provide more insight into the site energies of both PheoD1 and PheoD2 (including the corresponding Qx transitions, which are often claimed to be degenerate at 543 nm) and to attest that the above two assignments are most likely incorrect, we studied a large number of isolated RC preparations from spinach and wild-type Chlamydomonas reinhardtii (at different levels of intactness) as well as the Chlamydomonas reinhardtii mutant (D2-L209H), in which the active branch PheoD1 is genetically replaced with chlorophyll a (Chl a). We show that the Qx-/Qy-region site-energies of PheoD1 and PheoD2 are ~545/680 nm and ~541.5/670 nm, respectively, in good agreement with our previous assignment [Jankowiak et al. J. Phys. Chem. B 2002, 106, 8803–8814]. The latter values should be used to model excitonic structure and excitation energy transfer dynamics of the PSII RCs.Partial support to B.N. (involved in calculations) was provided by the NSF EPSCoR Grant. V.Z. (involved in writing the manuscript) acknowledges support by NSERC. R.T.S., R.P., and M.S. were involved in the design and preparation of D2-mutant and RCs. They acknowledge support from USDOE, Photosynthetic Antennae Research Center (R.T.S.), MICIN (Grant AGL2008-00377) in Spain (R.P.), and the U.S. Department of Energy’s Photosynthetic Systems Program within the Chemical Sciences, Geosciences, and Biosciences Division of the Office of Basic Energy Sciences under NREL Contract #DE-AC36-08-GO28308 (M.S.).Peer reviewe