Copper effect on cytochrome b559 of photosystem II under photoinhibitory conditions

The definitive version is available at www.blackwell-synergy.comToxic Cu(II) effect on Cytochrome b559 under aerobic photoinhibitory conditions was examined in two different PSII membrane preparations active in oxygen evolution. The preparations differ in the content of Cytochrome b559 redox potential forms. Difference absorption spectra showed that the presence of Cu(II) induced the oxidation of the high-potential form of Cytochrome b559 in the dark. Addition of hydroquinone reduced the total oxidised high-potential form of Cytochrome b559 present in Cu(II)-treated PSII membranes indicating that no conversion to the low-potential form took place. Spectroscopic determinations of Cytochrome b559 during photoinhibitory treatment showed slower kinetics of Cu(II) effect on Cytochrome b559 as compared to the rapid loss of oxygen evolution activity in the same conditions. This result indicates that Cytochrome b559 is affected after PSII centers are photoinhibited. The high-potential form was more sensitive to toxic Cu(II) action than the low-potential form under illumination at pH 6.0. The content of the high-potential form of Cytochrome b559 was completely lost, however the low-potential content was unaffected in these conditions. This loss did not involve cytochrome protein degradation. Results are discussed in terms of different binding properties of the heme iron to the protonated or unprotonated histidine ligand in the high-potential and low-potential forms of Cytochrome b559, respectively.M. Bernal was recipient of an I3P Programme fellowship from Consejo Superior de Investigaciones Científicas. This work was supported by the Dirección General de Investigación (Grant BMC2002-00031) to R.P. and Gobierno de Aragón (Grant P015/2001) to I.Y., and it has been done within GC DGA 2002 Program of Gobierno de Aragón.Peer reviewe

Changes in photosynthetic electron transfer and state transitions in an herbicide-resistant D1 mutant from soybean cell cultures

The definitive version is available at: http://www.sciencedirect.com/science//journal/00052728Anomalies in photosynthetic activity of the soybean cell line STR7, carrying a single mutation (S268P) in the chloroplastic gene psbA that codes for the D1 protein of the photosystem II, have been examined using different spectroscopic techniques. Thermoluminescence emission experiments have shown important differences between STR7 mutant and wild type cells. The afterglow band induced by both white light flashes and far-red continuous illumination was downshifted by about 4 °C and the Q band was upshifted by 5 °C. High temperature thermoluminescence measurements suggested a higher level of lipid peroxidation in mutant thylakoid membranes. In addition, the reduction rate of P700+ was significantly accelerated in STR7 suggesting that the mutation led to an activation of the photosystem I cyclic electron flow. Modulated fluorescence measurements performed at room temperature as well as fluorescence emission spectra at 77 K revealed that the STR7 mutant is defective in state transitions. Here, we discuss the hypothesis that activation of the cyclic electron flow in STR7 cells may be a mechanism to compensate the reduced activity of photosystem II caused by the mutation. We also propose that the impaired state transitions in the STR7 cells may be due to alterations in thylakoid membrane properties induced by a low content of unsaturated lipids.This work was supported by grants from the Ministry of Education and Culture of Spain (BFU-BMC2004-04914-C02-01, BMC2002-00031 and BFU-BMC2005-07422-C02-01) and Andalusia Government (PAI CVI-261).Peer reviewe

The photosynthetic cytochrome c 550 from the diatom Phaeodactylum tricornutum

The photosynthetic cytochrome c550 from the marine diatom Phaeodactylum tricornutum has been purified and characterized. Cytochrome c550 is mostly obtained from the soluble cell extract in relatively large amounts. In addition, the protein appeared to be truncated in the last hydrophobic residues of the C-terminus, both in the soluble cytochrome c550 and in the protein extracted from the membrane fraction, as deduced by mass spectrometry analysis and the comparison with the gene sequence. Interestingly, it has been described that the C-terminus of cytochrome c550 forms a hydrophobic finger involved in the interaction with photosystem II in cyanobacteria. Cytochrome c550 was almost absent in solubilized photosystem II complex samples, in contrast with the PsbO and Psb31 extrinsic subunits, thus suggesting a lower affinity of cytochrome c550 for the photosystem II complex. Under iron-limiting conditions the amount of cytochrome c550 decreases up to about 45% as compared to iron-replete cells, pointing to an iron-regulated synthesis. Oxidized cytochrome c550 has been characterized using continuous wave EPR and pulse techniques, including HYSCORE, and the obtained results have been interpreted in terms of the electrostatic charge distribution in the surroundings of the heme centre.This work was supported by the Spanish Ministry of Economy and Competitiveness (BIO2012-35271, BIO2015-64169-P, MAT2011-23861 and CTQ2015-64486-R) the Andalusian Government (PAIDI BIO-022) and the Aragón Government (Grupo consolidado B-18). All these grants were partially financed by the EU FEDER ProgramPeer 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

The singular properties of photosynthetic cytochrome c 550 from the diatom Phaeodactylum tricornutum suggest new alternative functions

Cytochrome c 550 is an extrinsic component in the luminal side of photosystem II (PSII) in cyanobacteria, as well as in eukaryotic algae from the red photosynthetic lineage including, among others, diatoms. We have established that cytochrome c 550 from the diatom Phaeodactylum tricornutum can be obtained as a complete protein from the membrane fraction of the alga, although a C-terminal truncated form is purified from the soluble fractions of this diatom as well as from other eukaryotic algae. Eukaryotic cytochromes c 550 show distinctive electrostatic features as compared with cyanobacterial cytochrome c 550 . In addition, co-immunoseparation and mass spectrometry experiments, as well as immunoelectron microscopy analyses, indicate that although cytochrome c 550 from P. tricornutum is mainly located in the thylakoid domain of the chloroplast – where it interacts with PSII –, it can also be found in the chloroplast pyrenoid, related with proteins linked to the CO 2 concentrating mechanism and assimilation. These results thus suggest new alternative functions of this heme protein in eukaryotes.Ministerio de Economía, Industria y Competitividad BIO2015-64169-PJunta de Andalucía PAIDI BIO-02

The photosynthetic cytochrome c550 from the diatom Phaeodactylum tricornutum

The photosynthetic cytochrome c550 from the marine diatom Phaeodactylum tricornutum has been purified and characterized. Cytochrome c550 is mostly obtained from the soluble cell extract in relatively large amounts. In addition, the protein appeared to be truncated in the last hydrophobic residues of the C-terminus, both in the soluble cytochrome c550 and in the protein extracted from the membrane fraction, as deduced by mass spectrometry analysis and the comparison with the gene sequence. Interestingly, it has been described that the C-terminus of cytochrome c550 forms a hydrophobic finger involved in the interaction with photosystem II in cyanobacteria. Cytochrome c550 was almost absent in solubilized photosystem II complex samples, in contrast with the PsbO and Psb31 extrinsic subunits, thus suggesting a lower affinity of cytochrome c550 for the photosystem II complex. Under iron-limiting conditions the amount of cytochrome c550 decreases up to about 45% as compared to iron-replete cells, pointing to an iron-regulated synthesis. Oxidized cytochrome c550 has been characterized using continuous wave EPR and pulse techniques, including HYSCORE, and the obtained results have been interpreted in terms of the electrostatic charge distribution in the surroundings of the heme centre

Thermoluminescence as a complementary technique for the toxicological evaluation of chemicals in photosynthetic organisms

© 2014. Thermoluminescence is a simple technique very useful for studying electron transfer reactions on photosystem II (standard thermoluminescence) or the level of lipid peroxidation in membranes (high temperature thermoluminescence) in photosynthetic organisms. Both techniques were used to investigate the effects produced on Chlorella vulgaris cells by six compounds: the chemical intermediates bromobenzene and diethanolamine, the antioxidant propyl gallate, the semiconductor indium nitrate, the pesticide sodium monofluoroacetate and the antimalarial drug chloroquine. Electron transfer activity of the photosystem II significantly decreased after the exposure of Chlorella cells to all the six chemicals used. Lipid peroxidation was slightly decreased by the antioxidant propyl gallate, not changed by indium nitrate and very potently stimulated by diethanolamine, chloroquine, sodium monofluoroacetate and bromobenzene. For five of the chemicals studied (not bromobenzene) there is a very good correlation between the cytotoxic effects in Chlorella cells measured by the algal growth inhibition test, and the inhibition of photosystem II activity. The results suggest that one very important effect of these chemicals in Chlorella cells is the inhibition of photosynthetic metabolism by the blocking of photosystem II functionality. In the case of sodium monofluoroacetate, diethanolamine and chloroquine this inhibition seems to be related with the induction of high level of lipid peroxidation in cells that may alter the stability of photosystem II. The results obtained by both techniques supply information that can be used as a supplement to the growth inhibition test and allows a more complete assessment of the effects of a chemical in photosynthetic organisms of aquatic ecosystems.Peer Reviewe

Copper effect on cytochrome b559 of photosystem II under photoinhibitory conditions

The definitive version is available at www.blackwell-synergy.comToxic Cu(II) effect on Cytochrome b559 under aerobic photoinhibitory conditions was examined in two different PSII membrane preparations active in oxygen evolution. The preparations differ in the content of Cytochrome b559 redox potential forms. Difference absorption spectra showed that the presence of Cu(II) induced the oxidation of the high-potential form of Cytochrome b559 in the dark. Addition of hydroquinone reduced the total oxidised high-potential form of Cytochrome b559 present in Cu(II)-treated PSII membranes indicating that no conversion to the low-potential form took place. Spectroscopic determinations of Cytochrome b559 during photoinhibitory treatment showed slower kinetics of Cu(II) effect on Cytochrome b559 as compared to the rapid loss of oxygen evolution activity in the same conditions. This result indicates that Cytochrome b559 is affected after PSII centers are photoinhibited. The high-potential form was more sensitive to toxic Cu(II) action than the low-potential form under illumination at pH 6.0. The content of the high-potential form of Cytochrome b559 was completely lost, however the low-potential content was unaffected in these conditions. This loss did not involve cytochrome protein degradation. Results are discussed in terms of different binding properties of the heme iron to the protonated or unprotonated histidine ligand in the high-potential and low-potential forms of Cytochrome b559, respectively.M. Bernal was recipient of an I3P Programme fellowship from Consejo Superior de Investigaciones Científicas. This work was supported by the Dirección General de Investigación (Grant BMC2002-00031) to R.P. and Gobierno de Aragón (Grant P015/2001) to I.Y., and it has been done within GC DGA 2002 Program of Gobierno de Aragón.Peer reviewe

Reconstitution, spectroscopy, and redox properties of the photosynthetic recombinant cytochrome b559 from higher plants

El pdf del artículo es la versión pre-print.A study of the in vitro reconstitution of sugar beet cytochrome b 559 of the photosystem II is described. Both α and β cytochrome subunits were first cloned and expressed in Escherichia coli. In vitro reconstitution of this cytochrome was carried out with partially purified recombinant subunits from inclusion bodies. Reconstitution with commercial heme of both (αα) and (ββ) homodimers and (αβ) heterodimer was possible, the latter being more efficient. The absorption spectra of these reconstituted samples were similar to that of the native heterodimer cytochrome b 559 form. As shown by electron paramagnetic resonance and potentiometry, most of the reconstituted cytochrome corresponded to a low spin form with a midpoint redox potential +36 mV, similar to that from the native purified cytochrome b 559. Furthermore, during the expression of sugar beet and Synechocystis sp. PCC 6803 cytochrome b 559 subunits, part of the protein subunits were incorporated into the host bacterial inner membrane, but only in the case of the β subunit from the cyanobacterium the formation of a cytochrome b 559-like structure with the bacterial endogenous heme was observed. The reason for that surprising result is unknown. This in vivo formed (ββ) homodimer cytochrome b 559-like structure showed similar absorption and electron paramagnetic resonance spectral properties as the native purified cytochrome b 559. A higher midpoint redox potential (+126 mV) was detected in the in vivo formed protein compared to the in vitro reconstituted form, most likely due to a more hydrophobic environment imposed by the lipid membrane surrounding the heme.This work was supported by Grants AGL2008-00377, MAT2008-03461, and BFU2007-68107-C02-01 from the Spanish Ministry of Science and Innovation (MICINN), PADI CVI-261 from the Andalusia Regional Government, and DGA-GC E33 and DGA-GE B18 from Aragon Regional Government. All these Grants were partially financed by the EU FEDER Program. M. A. Luján would like to thank the FPI Fellowship Program of the MICINN for financial support.Peer reviewe

Detergent effect on cytochrome b559 electron paramagnetic resonance signals in the photosystem II reaction centre

The definitive version is available at: http://www.rsc.org/publishing/journals/PP/The detergent effect on Cytochrome b559 from spinach photosystem II was studied by electron paramagnetic resonance (EPR) spectroscopy in D1–D2–Cyt b559 complex preparations. Various n-dodecyl--D-maltoside concentrations from 0 to 0.2%(w/v) were used to stabilise the D1–D2–Cyt b559 complexes. Low spin heme EPR spectra were obtained but the gz feature positions changed depending on the detergent conditions. Redox potentiometric titrations showed a unique redox potential cytochrome b559 form (Em=+123–150 mV) in all the D1–D2–Cyt b559 complex preparations indicating that detergent does not affect this property of the protein in those conditions. A similar effect on Cytochrome b559 EPR spectrum was observed in more intact photosystem II preparations independently of their aggregation state. This finding indicates that changes due to detergent could be a common phenomenon in photosystem II complexes. Results are discussed in terms of the environment each detergent provides to the protein.I. G.-R. was recipient of a fellowship from the Ministry of Education and Culture of Spain. This work was supported by the Dirección General de Investigación Científica y Técnica (Grants PB98-1632 to R.P. and PB97-1135 to J.M.O.) and by the Diputación General de Aragón (Projects P17/98 to P.J.A. and P111/2001 to J.I.M.).Peer reviewe