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1.
Photosynth Res ; 142(3): 307-319, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31482263

RESUMO

A kinetic-LED-array-spectrophotometer (Klas) was recently developed for measuring in vivo redox changes of P700, plastocyanin (PCy), and ferredoxin (Fd) in the near-infrared (NIR). This spectrophotometer is used in the present work for in vitro light-induced measurements with various combinations of photosystem I (PSI) from tobacco and two different cyanobacteria, spinach plastocyanin, cyanobacterial cytochrome c6 (cyt. c6), and Fd. It is shown that cyt. c6 oxidation contributes to the NIR absorption changes. The reduction of (FAFB), the terminal electron acceptor of PSI, was also observed and the shape of the (FAFB) NIR difference spectrum is similar to that of Fd. The NIR difference spectra of the electron-transfer cofactors were compared between different organisms and to those previously measured in vivo, whereas the relative absorption coefficients of all cofactors were determined by using single PSI turnover conditions. Thus, the (840 nm minus 965 nm) extinction coefficients of the light-induced species (oxidized minus reduced for PC and cyt. c6, reduced minus oxidized for (FAFB), and Fd) were determined with values of 0.207 ± 0.004, - 0.033 ± 0.006, - 0.036 ± 0.008, and - 0.021 ± 0.005 for PCy, cyt. c6, (FAFB) (single reduction), and Fd, respectively, by taking a reference value of + 1 for P700+. The fact that the NIR P700 coefficient is larger than that of PCy and much larger than that of other contributing species, combined with the observed variability in the NIR P700 spectral shape, emphasizes that deconvolution of NIR signals into different components requires a very precise determination of the P700 spectrum.


Assuntos
Proteínas de Bactérias/química , Complexo de Proteína do Fotossistema I/metabolismo , Proteínas de Plantas/química , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Proteínas de Bactérias/metabolismo , Citocromos c6/química , Citocromos c6/metabolismo , Transporte de Elétrons , Ferredoxinas/metabolismo , Oxirredução , Complexo de Proteína do Fotossistema I/química , Proteínas de Plantas/metabolismo , Plastocianina/química , Plastocianina/metabolismo , Espectrofotometria Ultravioleta , Espectroscopia de Luz Próxima ao Infravermelho/instrumentação , Spinacia oleracea/química , Synechocystis/química , Tabaco/química
2.
Nanotoxicology ; 13(4): 447-454, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30704326

RESUMO

The molecular mechanisms behind the adaptive responses for interactions between organisms and nanoparticles, such as silver nanoparticles (AgNPs), are of great concern. In this study, the transcriptome of freshwater alga Chlamydomonas reinhardtii was characterized via RNA sequencing (RNA-seq) after exposure to a nontoxic concentration of AgNPs (0.5 mg/L). The cytochrome c6 (CYC6) and ferredoxin-5 (FDX5) genes were identified with the greatest increase in expression level, which were indications of the copper deficiency in the algae. Gene set enrichment analysis also showed significant enrichment of copper deficiency responsive genes in the transcriptome of algae exposed to AgNPs. These results indicated that AgNPs induced a copper deficiency response in algae, and the excessive intracellular copper content suggested this was due to functional copper deficiency. This deficiency response was further validated to be regulated by transcription factor CRR1 (copper response regulator 1) according to the assays on the mutant strain with defect of CRR1. To the best of our knowledge, this is the first corroboration of a CRR1-targeted copper deficiency response in algae following AgNP exposure. Given the function of copper in fundamental metabolic pathways, such as photosynthesis and respiration, we propose a potential role of CRR1-targeted copper deficiency as an adaptation of algae after exposure to AgNPs.


Assuntos
Chlamydomonas reinhardtii/efeitos dos fármacos , Cobre/deficiência , Nanopartículas Metálicas/toxicidade , Oxirredutases/genética , Proteínas de Plantas/genética , Prata/toxicidade , Sequência de Bases , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Citocromos c6/genética , Ferredoxinas/genética , Fotossíntese/efeitos dos fármacos , Fotossíntese/genética , Transcriptoma/efeitos dos fármacos
3.
Biochim Biophys Acta Bioenerg ; 1860(1): 60-68, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30414412

RESUMO

Cytochrome c6 is a soluble electron carrier, present in all known cyanobacteria, that has been replaced by plastocyanin in plants. Despite their high structural differences, both proteins have been reported to be isofunctional in cyanobacteria and green algae, acting as alternative electron carriers from the cytochrome b6-f complex to photosystem I or terminal oxidases. We have investigated the subcellular localization of both cytochrome c6 and plastocyanin in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 grown in the presence of combined nitrogen and under diazotrophic conditions. Our studies conclude that cytochrome c6 is expressed at significant levels in heterocysts, even in the presence of copper, condition in which it is strongly repressed in vegetative cells. However, the copper-dependent regulation of plastocyanin is not altered in heterocysts. In addition, in heterocysts, cytochrome c6 has shown to be the main soluble electron carrier to cytochrome c oxidase-2 in respiration. A cytochrome c6 deletion mutant is unable to grow under diazotrophic conditions in the presence of copper, suggesting that cytochrome c6 plays an essential role in the physiology of heterocysts that cannot be covered by plastocyanin.


Assuntos
Anabaena/fisiologia , Respiração Celular , Citocromos c6/fisiologia , Fotossíntese , Cobre/farmacologia , Cianobactérias , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Fixação de Nitrogênio , Plastocianina/fisiologia
4.
Mol Biol Rep ; 45(6): 1745-1758, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30159639

RESUMO

Cytochromes are important components of photosynthetic electron transport chain. Here we report on genetic transformation of Cytochrome c6 (UfCyt c6) gene from Ulva fasciata Delile in tobacco for enhanced photosynthesis and growth. UfCyt c6 cDNA had an open reading frame of 330 bp encoding a polypeptide of 109 amino acids with a predicted molecular mass of 11.65 kDa and an isoelectric point of 5.21. UfCyt c6 gene along with a tobacco petE transit peptide sequence under control of CaMV35S promoter was transformed in tobacco through Agrobacterium mediated genetic transformation. Transgenic tobacco grew normal and exhibited enhanced growth as compared to wild type (WT) and vector control (VC) tobacco. Transgenic tobacco had higher contents of photosynthetic pigments and better ratios of photosynthetic pigments. The tobacco expressing UfCyt c6 gene exhibited higher photosynthetic rate and improved water use efficiency. Further activity of the water-splitting complex, photosystem II quantum yield, photochemical quenching, electron transfer rate, and photosynthetic yield were found comparatively higher in transgenic tobacco as compared to WT and VC tobacco. Alternatively basal quantum yield of non-photochemical processes in PSII and non-photochemical quenching were estimated lower in tobacco expressing UfCyt c6 gene. As a result of improved photosynthetic performance the transgenic tobacco had higher contents of sugar and starch, and exhibited comparatively better growth. To the best of our knowledge this is the first report on expression of UfCyt c6 gene from U. fasciata for improved photosynthesis and growth in tobacco.


Assuntos
Citocromos c6/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Clorofila/metabolismo , Citocromos c6/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Oxirredução , Fotossíntese/genética , Complexo de Proteína do Fotossistema II/genética , Proteínas de Plantas/genética , Alga Marinha/metabolismo , Tilacoides/metabolismo , Tabaco/genética , Tabaco/metabolismo , Transfecção/métodos , Ulva/metabolismo
5.
J Biol Chem ; 293(23): 9090-9100, 2018 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-29695502

RESUMO

The binding of photosystem I (PS I) from Thermosynechococcus elongatus to the native cytochrome (cyt) c6 and cyt c from horse heart (cyt cHH) was analyzed by oxygen consumption measurements, isothermal titration calorimetry (ITC), and rigid body docking combined with electrostatic computations of binding energies. Although PS I has a higher affinity for cyt cHH than for cyt c6, the influence of ionic strength and pH on binding is different in the two cases. ITC and theoretical computations revealed the existence of unspecific binding sites for cyt cHH besides one specific binding site close to P700 Binding to PS I was found to be the same for reduced and oxidized cyt cHH Based on this information, suitable conditions for cocrystallization of cyt cHH with PS I were found, resulting in crystals with a PS I:cyt cHH ratio of 1:1. A crystal structure at 3.4-Å resolution was obtained, but cyt cHH cannot be identified in the electron density map because of unspecific binding sites and/or high flexibility at the specific binding site. Modeling the binding of cyt c6 to PS I revealed a specific binding site where the distance and orientation of cyt c6 relative to P700 are comparable with cyt c2 from purple bacteria relative to P870 This work provides new insights into the binding modes of different cytochromes to PS I, thus facilitating steps toward solving the PS I-cyt c costructure and a more detailed understanding of natural electron transport processes.


Assuntos
Proteínas de Bactérias/metabolismo , Cianobactérias/metabolismo , Citocromos c6/metabolismo , Citocromos c/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Animais , Proteínas de Bactérias/química , Sítios de Ligação , Cianobactérias/química , Citocromos c/química , Citocromos c6/química , Cavalos , Simulação de Acoplamento Molecular , Concentração Osmolar , Complexo de Proteína do Fotossistema I/química , Eletricidade Estática
6.
FEBS Lett ; 590(20): 3639-3648, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27685247

RESUMO

Plastocyanin (petE) plays an essential role in photosynthesis as an electron carrier between cytochrome b6 f and photosystem I, and in some cyanobacteria it can be replaced by the haem-containing protein, cytochrome c6 (petJ). In Synechocystis sp. PCC 6803, transcription of petE and petJ is activated and repressed, respectively, by Cu. Here, we show that Ni can act similarly to Cu in inducing petE and repressing petJ, thus leading to a partial switch between cytochrome c6 and plastocyanin. Transcription of these genes is only altered by Ni in Cu-depleted medium, and none of the Ni-dependent transcription factors described in Synechocystis, NrsR and InrS seem to be involved in this regulation. Finally, we show that plastocyanin is essential for growth under conditions of excess Ni.


Assuntos
Citocromos c6/genética , Níquel/metabolismo , Plastocianina/genética , Synechocystis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Citocromos c6/metabolismo , Regulação Bacteriana da Expressão Gênica , Plastocianina/metabolismo , Synechocystis/genética , Synechocystis/metabolismo , Transcrição Genética
7.
Photosynth Res ; 127(1): 5-11, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25527460

RESUMO

The quantum yield for light-induced H2 generation was measured for a previously optimized bio-hybrid cytochrome c 6-crosslinked PSI(C13G)-1,8-octanedithiol-[FeFe]-H2ase(C97G) (PSI-H2ase) nanoconstruct. The theoretical quantum yield for the PSI-H2ase nanoconstruct is 0.50 molecules of H2 per photon absorbed, which equates to a requirement of two photons per H2 generated. Illumination of the PSI-H2ase nanoconstruct with visible light between 400 and 700 nm resulted in an average quantum yield of 0.10-0.15 molecules of H2 per photon absorbed, which equates to a requirement of 6.7-10 photons per H2 generated. A possible reason for the difference between the theoretical and experimental quantum yield is the occurrence of non-productive PSI(C13G)-1,8-octanedithiol-PSIC13G (PSI-PSI) conjugates, which would absorb light without generating H2. Assuming the thiol-Fe coupling is equally efficient at producing PSI-PSI conjugates as well as in producing PSI-H2ase nanoconstructs, the theoretical quantum yield would decrease to 0.167 molecules of H2 per photon absorbed, which equates to 6 photons per H2 generated. This value is close to the range of measured values in the current study. A strategy that purifies the PSI-H2ase nanoconstructs from the unproductive PSI-PSI conjugates or that incorporates different chemistries on the PSI and [FeFe]-H2ase enzyme sites could potentially allow the PSI-H2ase nanoconstruct to approach the expected theoretical quantum yield for light-induced H2 generation.


Assuntos
Hidrogênio/metabolismo , Nanoestruturas/química , Complexo de Proteína do Fotossistema I/metabolismo , Biocombustíveis , Reagentes para Ligações Cruzadas/química , Citocromos c6/química , Citocromos c6/metabolismo , Hidrogenase/química , Hidrogenase/metabolismo , Ferro/química , Ferro/metabolismo , Luz , Complexo de Proteína do Fotossistema I/química , Teoria Quântica , Compostos de Sulfidrila/química
8.
Biochim Biophys Acta ; 1847(12): 1549-59, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26407632

RESUMO

In the Phaeodactylum tricornutum alga, as in most diatoms, cytochrome c6 is the only electron donor to photosystem I, and thus they lack plastocyanin as an alternative electron carrier. We have investigated, by using laser-flash absorption spectroscopy, the electron transfer to Phaeodactylum photosystem I from plastocyanins from cyanobacteria, green algae and plants, as compared with its own cytochrome c6. Diatom photosystem I is able to effectively react with eukaryotic acidic plastocyanins, although with less efficiency than with Phaeodactylum cytochrome c6. This efficiency, however, increases in some green alga plastocyanin mutants mimicking the electrostatics of the interaction site on the diatom cytochrome. In addition, the structure of the transient electron transfer complex between cytochrome c6 and photosystem I from Phaeodactylum has been analyzed by computational docking and compared to that of green lineage and mixed systems. Taking together, the results explain why the Phaeodactylum system shows a lower efficiency than the green systems, both in the formation of the properly arranged [cytochrome c6-photosystem I] complex and in the electron transfer itself.


Assuntos
Citocromos c6/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Plastocianina/metabolismo , Estramenópilas/metabolismo , Citocromos c6/química , Cinética , Simulação de Acoplamento Molecular , Fotossíntese , Complexo de Proteína do Fotossistema I/química , Plastocianina/química , Ligação Proteica , Estramenópilas/fisiologia
9.
PLoS One ; 10(6): e0129081, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26052941

RESUMO

Ferroproteins arose early in Earth's history, prior to the emergence of oxygenic photosynthesis and the subsequent reduction of bioavailable iron. Today, iron availability limits primary productivity in about 30% of the world's oceans. Diatoms, responsible for nearly half of oceanic primary production, have evolved molecular strategies for coping with variable iron concentrations. Our understanding of the evolutionary breadth of these strategies has been restricted by the limited number of species for which molecular sequence data is available. To uncover the diversity of strategies marine diatoms employ to meet cellular iron demands, we analyzed 367 newly released marine microbial eukaryotic transcriptomes, which include 47 diatom species. We focused on genes encoding proteins previously identified as having a role in iron management: iron uptake (high-affinity ferric reductase, multi-copper oxidase, and Fe(III) permease); iron storage (ferritin); iron-induced protein substitutions (flavodoxin/ferredoxin, and plastocyanin/cytochrome c6) and defense against reactive oxygen species (superoxide dismutases). Homologs encoding the high-affinity iron uptake system components were detected across the four diatom Classes suggesting an ancient origin for this pathway. Ferritin transcripts were also detected in all Classes, revealing a more widespread utilization of ferritin throughout diatoms than previously recognized. Flavodoxin and plastocyanin transcripts indicate possible alternative redox metal strategies. Predicted localization signals for ferredoxin identify multiple examples of gene transfer from the plastid to the nuclear genome. Transcripts encoding four superoxide dismutase metalloforms were detected, including a putative nickel-coordinating isozyme. Taken together, our results suggest that the majority of iron metabolism genes in diatoms appear to be vertically inherited with functional diversity achieved via possible neofunctionalization of paralogs. This refined view of iron use strategies in diatoms elucidates the history of these adaptations, and provides potential molecular markers for determining the iron nutritional status of different diatom species in environmental samples.


Assuntos
Diatomáceas/genética , Diatomáceas/metabolismo , Evolução Molecular , Variação Genética , Ferro/metabolismo , Sequência de Aminoácidos , Citocromos c6/metabolismo , Diatomáceas/enzimologia , Ferredoxinas/química , Ferredoxinas/genética , Ferritinas/genética , Flavodoxina/genética , Dados de Sequência Molecular , Oxirredução , Filogenia , Fitoplâncton/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Superóxido Dismutase/metabolismo , Transcrição Genética , Ubiquitina/metabolismo
10.
Biofizika ; 60(2): 270-92, 2015.
Artigo em Russo | MEDLINE | ID: mdl-26016024

RESUMO

The application of Brownian dynamics for simulation of transient protein-protein interactions is reviewed. The review focuses on theoretical basics of Brownian dynamics method, its particular implementations, advantages and drawbacks of the method. The outlook for future development of Brownian dynamics-based simulation techniques is discussed. Special attention is given to analysis of Brownian dynamics trajectories. The second part of the review is dedicated to the role of Brownian dynamics simulations in studying photosynthetic electron transport. Interactions of mobile electron carriers (plastocyanin, cytochrome c6, and ferredoxin) with their reaction partners (cytochrome b6f complex, photosystem I, ferredoxin:NADP-reductase, and hydrogenase) are considered.


Assuntos
Fenômenos Biofísicos , Citocromos c6/química , Fotossíntese , Plastocianina/química , Citocromos f , Transporte de Elétrons , Ferredoxinas/química , Cinética , Modelos Moleculares , Simulação de Dinâmica Molecular , Complexo de Proteína do Fotossistema I , Conformação Proteica
11.
Mol Microbiol ; 96(6): 1298-317, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25825009

RESUMO

The microaerophilic food-borne pathogen Campylobacter jejuni uses complex cytochrome-rich respiratory chains for growth and host colonisation. Cytochrome c biogenesis requires haem ligation to reduced apocytochrome cysteines, catalysed by the cytochrome c synthase, CcsBA. While ccsBA could not be deleted, we showed that the thiol reductase DsbD and the CcsX homologue Cj1207 are involved in, but not essential for, cytochromes c biogenesis. Mutant phenotypic analyses and biochemical studies with purified proteins revealed that the mono-haem c-type cytochromes Cj1153 (CccA) and Cj1020 (CccB) and the di-haem Cj0037 (CccC) are electron donors to the cb-oxidase (CcoNOQP), with CccC being more efficient than CccA. Remarkably, cccA deletion or site-directed mutagenesis resulted in an almost complete loss of all other c-type cytochromes. Cytochrome c structural and biogenesis genes were still transcribed in the cccA deletion mutant and the quinol oxidase genes (cioAB) were up-regulated. Cytochrome c production could be rescued in this mutant by growth with exogenous dithiothreitol or L-cysteine, suggesting that in the absence of CccA, apocytochrome c haem binding motifs become oxidised, preventing haem attachment. Our results identify CccA, the most abundant periplasmic c-type cytochrome in C. jejuni, as a novel and unexpected protein required for cytochrome c biogenesis in this pathogen.


Assuntos
Campylobacter jejuni/metabolismo , Citocromos c6/metabolismo , Citocromos c/biossíntese , Proteínas de Bactérias/metabolismo , Campylobacter jejuni/genética , Cisteína/metabolismo , Citocromos c/genética , Transporte de Elétrons , Escherichia coli/genética , Heme/metabolismo , Mutagênese Sítio-Dirigida , Oxirredutases/metabolismo , Compostos de Sulfidrila/metabolismo
12.
Proc Natl Acad Sci U S A ; 112(9): 2644-51, 2015 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-25646490

RESUMO

Inorganic elements, although required only in trace amounts, permit life and primary productivity because of their functions in catalysis. Every organism has a minimal requirement of each metal based on the intracellular abundance of proteins that use inorganic cofactors, but elemental sparing mechanisms can reduce this quota. A well-studied copper-sparing mechanism that operates in microalgae faced with copper deficiency is the replacement of the abundant copper protein plastocyanin with a heme-containing substitute, cytochrome (Cyt) c6. This switch, which is dependent on a copper-sensing transcription factor, copper response regulator 1 (CRR1), dramatically reduces the copper quota. We show here that in a situation of marginal copper availability, copper is preferentially allocated from plastocyanin, whose function is dispensable, to other more critical copper-dependent enzymes like Cyt oxidase and a ferroxidase. In the absence of an extracellular source, copper allocation to Cyt oxidase includes CRR1-dependent proteolysis of plastocyanin and quantitative recycling of the copper cofactor from plastocyanin to Cyt oxidase. Transcriptome profiling identifies a gene encoding a Zn-metalloprotease, as a candidate effecting copper recycling. One reason for the retention of genes encoding both plastocyanin and Cyt c6 in algal and cyanobacterial genomes might be because plastocyanin provides a competitive advantage in copper-depleted environments as a ready source of copper.


Assuntos
Chlamydomonas/metabolismo , Cobre/metabolismo , Consumo de Oxigênio/fisiologia , Fotossíntese/fisiologia , Ceruloplasmina/genética , Ceruloplasmina/metabolismo , Chlamydomonas/genética , Citocromos c6/genética , Citocromos c6/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Plastocianina/genética , Plastocianina/metabolismo
13.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 11): 2823-32, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25372674

RESUMO

The structure of cytochrome c6C from the mesophilic cyanobacterium Synechococcus sp. PCC 7002 has been determined at 1.03 Šresolution. This is the first structural report on the recently discovered cyanobacterial cytochrome c6-like proteins found in marine and nitrogen-fixing cyanobacteria. Despite high similarity in the overall three-dimensional fold between cytochromes c6 and c6C, the latter shows saliently different electrostatic properties in terms of surface charge distribution and dipole moments. Its midpoint redox potential is less than half of the value for typical c6 cytochromes and results mainly from the substitution of one residue in the haem pocket. Here, high-resolution crystal structures of mutants of both cytochromes c6 and c6C are presented, and the impact of the mutation of specific residues in the haem-binding pocket on the redox potential is discussed. These findings contribute to the elucidation of the structure-function relationship of c6-like cytochromes.


Assuntos
Proteínas de Bactérias/química , Citocromos c6/química , Heme/metabolismo , Synechococcus/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Citocromos c6/metabolismo , Modelos Moleculares , Oxirredução , Conformação Proteica , Synechococcus/metabolismo
14.
PLoS One ; 9(9): e108912, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25268225

RESUMO

Copper is an essential element involved in fundamental processes like respiration and photosynthesis. However, it becomes toxic at high concentration, which has forced organisms to control its cellular concentration. We have recently described a copper resistance system in the cyanobacterium Synechocystis sp. PCC 6803, which is mediated by the two-component system, CopRS, a RND metal transport system, CopBAC and a protein of unknown function, CopM. Here, we report the transcriptional responses to copper additions at non-toxic (0.3 µM) and toxic concentrations (3 µM) in the wild type and in the copper sensitive copR mutant strain. While 0.3 µM copper slightly stimulated metabolism and promoted the exchange between cytochrome c6 and plastocyanin as soluble electron carriers, the addition of 3 µM copper catalyzed the formation of ROS, led to a general stress response and induced expression of Fe-S cluster biogenesis genes. According to this, a double mutant strain copRsufR, which expresses constitutively the sufBCDS operon, tolerated higher copper concentration than the copR mutant strain, suggesting that Fe-S clusters are direct targets of copper toxicity in Synechocystis. In addition we have also demonstrated that InrS, a nickel binding transcriptional repressor that belong to the CsoR family of transcriptional factor, was involved in heavy metal homeostasis, including copper, in Synechocystis. Finally, global gene expression analysis of the copR mutant strain suggested that CopRS only controls the expression of copMRS and copBAC operons in response to copper.


Assuntos
Proteínas de Bactérias/genética , Cobre/toxicidade , Poluentes Ambientais/toxicidade , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Synechocystis/efeitos dos fármacos , Transcriptoma , Proteínas de Bactérias/metabolismo , Citocromos c6/genética , Citocromos c6/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Proteínas de Membrana Transportadoras/metabolismo , Anotação de Sequência Molecular , Mutação , Óperon , Plastocianina/genética , Plastocianina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Synechocystis/genética , Synechocystis/metabolismo
15.
Biochim Biophys Acta ; 1837(8): 1305-15, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24685428

RESUMO

The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b6f with the electron transfer proteins plastocyanin and cytochrome c6. A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c6 from the cyanobacterium Nostoc sp. PCC 7119 was studied using NMR spectroscopy and X-ray diffraction. The crystal structures of wild type, M58H and M58C cytochrome c6 were determined. The M58C variant is an excellent low potential mimic of the wild type protein and was used in chemical shift perturbation and paramagnetic relaxation NMR experiments to characterize the complex with cytochrome f. The interaction is highly dynamic and can be described as a pure encounter complex, with no dominant stereospecific complex. Ensemble docking calculations and Monte-Carlo simulations suggest a model in which charge-charge interactions pre-orient cytochrome c6 with its haem edge toward cytochrome f to form an ensemble of orientations with extensive contacts between the hydrophobic patches on both cytochromes, bringing the two haem groups sufficiently close to allow for rapid electron transfer. This model of complex formation allows for a gradual increase and decrease of the hydrophobic interactions during association and dissociation, thus avoiding a high transition state barrier that would slow down the dissociation process.


Assuntos
Citocromos c6/química , Citocromos f/química , Complexos Multiproteicos/química , Fotossíntese , Cianobactérias/química , Cianobactérias/metabolismo , Citocromos c6/metabolismo , Citocromos f/metabolismo , Transporte de Elétrons , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Método de Monte Carlo , Complexos Multiproteicos/metabolismo , Plastocianina/química , Plastocianina/metabolismo , Ligação Proteica , Conformação Proteica , Mapas de Interação de Proteínas , Difração de Raios X
16.
Methods Mol Biol ; 1132: 413-24, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24599871

RESUMO

The green unicellular alga Chlamydomonas reinhardtii has emerged as a very attractive model system for chloroplast genetic engineering. Algae can be transformed readily at the chloroplast level through bombardment of cells with a gene gun, and transformants can be selected using antibiotic resistance or phototrophic growth. An inducible chloroplast gene expression system could be very useful for several reasons. First, it could be used to elucidate the function of essential chloroplast genes required for cell growth and survival. Second, it could be very helpful for expressing proteins which are toxic to the algal cells. Third, it would allow for the reversible depletion of photosynthetic complexes thus making it possible to study their biogenesis in a controlled fashion. Fourth, it opens promising possibilities for hydrogen production in Chlamydomonas. Here we describe an inducible/repressible chloroplast gene expression system in Chlamydomonas in which the copper-regulated Cyc6 promoter drives the expression of the nuclear Nac2 gene encoding a protein which is targeted to the chloroplast where it acts specifically on the chloroplast psbD 5'-untranslated region and is required for the stable accumulation of the psbD mRNA and photosystem II. The system can be used for any chloroplast gene or transgene by placing it under the control of the psbD 5'-untranslated region.


Assuntos
Chlamydomonas reinhardtii/genética , Cloroplastos/genética , Transgenes/genética , Regiões 5' não Traduzidas/genética , Biolística/métodos , Cloroplastos/metabolismo , Cobre/farmacologia , Citocromos c6/genética , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Nucleotidiltransferases/genética , Complexo de Proteína do Fotossistema II/genética , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Proteínas Repressoras/genética , Transformação Genética
17.
Biochemistry ; 53(14): 2295-306, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24649965

RESUMO

The molecular wire-appended naphthoquinone 1-[15-(3-methyl-1,4-naphthoquinone-2-yl)]pentadecyl disulfide [(NQ(CH2)15S)2] has been incorporated into the A1A and A1B sites of Photosystem I (PS I) in the menB variant of Synechocystis sp. PCC 6803. Transient electron paramagnetic resonance studies show that the naphthoquinone headgroup displaces plastoquinone-9 from the A1A (and likely A1B) sites to a large extent. When a Pt nanoparticle is attached to the molecular wire by reductive cleavage of the disulfide and reaction with the resulting thiol, the PS I-NQ(CH2)15S-Pt nanoconstruct evolves dihydrogen at a rate of 67.3 µmol of H2 (mg of Chl)(-1) h(-1) [3.4 e(-) (PS I)(-1) s(-1)] after illumination for 1 h at pH 6.4. No dihydrogen is detected if wild-type PS I, which does not incorporate the quinone, is used or if either (NQ(CH2)15S)2 or the Pt nanoparticle is absent. Time-resolved optical studies of the PS I-NQ(CH2)15S-Pt nanoconstruct show that the lifetimes of the forward electron transfer to and reverse electron transfer from the iron-sulfur clusters are the same as in native PS I. Thus, electrons are not shuttled directly from the quinone to the Pt nanoparticle during either forward or reverse electron transfer. It is found that the rate of dihydrogen evolution in the PS I-NQ(CH2)15S-Pt nanoconstruct depends strongly on the concentration the sacrificial electron donor cytochrome c6. These observations can be explained if the iron-sulfur clusters are involved in stabilizing the electron; the ~50 ms residence time of the electron on FA or FB is sufficiently long to allow cytochrome c6 to reduce P700(+), thereby eliminating the recombination channel. In the absence of P700(+), slow electron transfer through the molecular wire to the Pt catalyst can occur, and hence, H2 evolution is observed.


Assuntos
Luz , Nanopartículas Metálicas , Naftoquinonas/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Platina/metabolismo , Sítios de Ligação , Citocromos c6/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Hidrogênio
18.
Biochem Biophys Res Commun ; 443(4): 1131-5, 2014 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-24216109

RESUMO

Cytochromes c are soluble electron carriers of relatively low molecular weight, containing single heme moiety. In cyanobacteria cytochrome c6 participates in electron transfer from cytochrome b6f complex to photosystem I. Recent phylogenetic analysis revealed the existence of a few families of proteins homologous to the previously mentioned. Cytochrome c6A from Arabidopsis thaliana was identified as a protein responsible for disulfide bond formation in response to intracellular redox state changes and c550 is well known element of photosystem II. However, function of cytochromes marked as c6B, c6C and cM as well as the physiological process in which they take a part still remain unidentified. Here we present the first structural and biophysical analysis of cytochrome from the c6B family from mesophilic cyanobacteria Synechococcus sp. WH 8102. Purified protein was crystallized and its structure was refined at 1.4 Å resolution. Overall architecture of this polypeptide resembles typical I-class cytochromes c. The main features, that distinguish described protein from cytochrome c6, are slightly red-shifted α band of UV-Vis spectrum as well as relatively low midpoint potential (113.2±2.2 mV). Although, physiological function of cytochrome c6B has yet to be determined its properties probably exclude the participation of this protein in electron trafficking between b6f complex and photosystem I.


Assuntos
Citocromos c6/química , Synechococcus/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Cristalografia por Raios X , Citocromos c6/genética , Heme/química , Ligação de Hidrogênio , Modelos Moleculares , Oxirredução , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Synechococcus/genética
19.
Biochemistry ; 52(48): 8687-95, 2013 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-24180741

RESUMO

Diatoms occupy a key branch in the evolutionary tree of oxygen-evolving photosynthetic organisms. Here, the electron transfer reaction mechanism from cytochrome c6 to photosystem I from the diatom Phaeodactylum tricornutum has been analyzed by laser-flash absorption spectroscopy. Kinetic traces of photosystem I reduction fit to biphasic curves, the analysis of the observed rate constants indicating that electron transfer occurs in a cytochrome c6/photosystem I transient complex, which undergoes a reorganization process from the initial encounter complex to the optimized final configuration. The mild ionic strength dependence of the rate constants makes evident the relatively weak electrostatically attractive nature of the interaction. Taken together, these results indicate that the "red" Phaeodactylum system is less efficient than "green" systems, both in the formation of the properly arranged (cytochrome c6/photosystem I) complex and in the electron transfer itself. The results obtained from cross-reactions with cytochrome c6 and photosystem I from cyanobacteria, green algae, and plants shed light on the different evolutionary pathway of the electron transfer to photosystem I in diatoms with regard to the way that it evolved in higher plants.


Assuntos
Diatomáceas/enzimologia , Complexo de Proteína do Fotossistema I/metabolismo , Arabidopsis/enzimologia , Clorófitas/enzimologia , Citocromos c6/metabolismo , Transporte de Elétrons/fisiologia , Cinética , Concentração Osmolar , Oxirredução , Fotossíntese/fisiologia
20.
Biochim Biophys Acta ; 1827(4): 529-39, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23416844

RESUMO

Intact fucoxanthin (Fucox)-chlorophyll (Chl)-binding protein I-photosystem I supercomplexes (FCPI-PSIs) were prepared by a newly developed simple fast procedure from centric diatoms Chaetoceros gracilis and Thalassiosira pseudonana to study the mechanism of their efficient solar energy accumulation. FCPI-PSI purified from C. gracilis contained 252 Chl a, 23 Chl c, 56 Fucox, 34 diadinoxanthin+diatoxanthin, 1 violaxanthin, 21 ß-carotene, and 2 menaquinone-4 per P700. The complex showed a high electron transfer activity at 185,000µmolmg Chl a(-1)·h(-1) to reduce methyl viologen from added cytochrome c6. We identified 14 and 21 FCP proteins in FCPI-PSI of C. gracilis and T. pseudonana, respectively, determined by N-terminal and internal amino acid sequences and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. PsaO and a red lineage Chla/b-binding-like protein (RedCAP), Thaps3:270215, were also identified. Severe detergent treatment of FCPI-PSI released FCPI-1 first, leaving the FCPI-2-PSI-core complex. FCPI-1 contained more Chl c and showed Chl a fluorescence at a shorter wavelength than FCPI-2, suggesting an excitation-energy transfer from FCPI-1 to FCPI-2 and then to the PSI core. Fluorescence emission spectra at 17K in FCPI-2 varied depending on the excitation wavelength, suggesting two independent energy transfer routes. We formulated a model of FCPI-PSI based on the biochemical assay results.


Assuntos
Proteínas de Ligação à Clorofila/metabolismo , Clorofila/metabolismo , Citocromos c6/metabolismo , Diatomáceas/metabolismo , Fragmentos de Peptídeos/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Clorofila/química , Clorofila A , Proteínas de Ligação à Clorofila/química , Cromatografia Líquida , Citocromos c6/química , Diatomáceas/citologia , Fluorescência , Fragmentos de Peptídeos/química , Fotoquímica , Complexo de Proteína do Fotossistema I/química , Espectrometria de Massas em Tandem
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