RESUMEN
The function of ascorbate peroxidase-related (APX-R) proteins, present in all green photosynthetic eukaryotes, remains unclear. This study focuses on APX-R from Chlamydomonas reinhardtii, namely, ascorbate peroxidase 2 (APX2). We showed that apx2 mutants exhibited a faster oxidation of the photosystem I primary electron donor, P700, upon sudden light increase and a slower re-reduction rate compared to the wild type, pointing to a limitation of plastocyanin. Spectroscopic, proteomic and immunoblot analyses confirmed that the phenotype was a result of lower levels of plastocyanin in the apx2 mutants. The redox state of P700 did not differ between wild type and apx2 mutants when the loss of function in plastocyanin was nutritionally complemented by growing apx2 mutants under copper deficiency. In this case, cytochrome c6 functionally replaces plastocyanin, confirming that lower levels of plastocyanin were the primary defect caused by the absence of APX2. Overall, the results presented here shed light on an unexpected regulation of plastocyanin level under copper-replete conditions, induced by APX2 in Chlamydomonas.
Asunto(s)
Ascorbato Peroxidasas , Chlamydomonas reinhardtii , Mutación , Plastocianina , Plastocianina/metabolismo , Plastocianina/genética , Ascorbato Peroxidasas/metabolismo , Ascorbato Peroxidasas/genética , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/genética , Cobre/metabolismo , Oxidación-Reducción , Complejo de Proteína del Fotosistema I/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Citocromos c6/metabolismo , Citocromos c6/genética , Proteómica/métodos , LuzRESUMEN
During photosynthesis, electrons are transferred between the cytochrome b6f complex and photosystem I. This is carried out by the protein plastocyanin in plant chloroplasts, or by either plastocyanin or cytochrome c6 in many cyanobacteria and eukaryotic algal species. There are three further cytochrome c6 homologs: cytochrome c6A in plants and green algae, and cytochromes c6B and c6C in cyanobacteria. The function of these proteins is unknown. Here, we present a comprehensive analysis of the evolutionary relationship between the members of the cytochrome c6 family in photosynthetic organisms. Our phylogenetic analyses show that cytochromes c6B and c6C are likely to be orthologs that arose from a duplication of cytochrome c6, but that there is no evidence for separate origins for cytochromes c6B and c6C. We therefore propose renaming cytochrome c6C as cytochrome c6B. We show that cytochrome c6A is likely to have arisen from cytochrome c6B rather than by an independent duplication of cytochrome c6, and present evidence for an independent origin of a protein with some of the features of cytochrome c6A in peridinin dinoflagellates. We conclude with a new comprehensive model of the evolution of the cytochrome c6 family which is an integral part of understanding the function of the enigmatic cytochrome c6 homologs.
Asunto(s)
Citocromos c6 , Citocromos/metabolismo , Citocromos c6/genética , Citocromos c6/metabolismo , Transporte de Electrón , Electrones , Fotosíntesis/genética , FilogeniaRESUMEN
Native cytochrome c6 was purified from an extract of strain BP-1 of the thermophilic cyanobacterium Thermosynechococcus elongatus. The protein was crystallized, and with only slight modifications of the buffer and vapour-diffusion conditions two different space groups were observed, namely H3 and C2. Both crystal structures were solved; they contained three and six molecules per asymmetric unit and were refined to 1.7 and 2.25â Å resolution, respectively. To date, the structure of native cytochrome c6 from T. elongatus has only been reported as a monomer using NMR spectroscopy, i.e. without addressing putative oligomerization, and related structures have only previously been solved using X-ray crystallography after recombinant gene overexpression in Escherichia coli. The reported space groups of related cyanobacterial cytochrome c6 structures differ from those reported here. Interestingly, the protein-protein interfaces that were observed utilizing X-ray crystallography could also explain homo-oligomerization in solution; specifically, trimerization is indicated by infra-red dynamic light scattering and blue native gel electrophoresis in solution. Trimers were also detected by mass spectrometry. Furthermore, there is an indication of post-translational methylation in the crystal structure. Additionally, the possibility of modifying the crystal size and the redox activity in the context of photosynthesis is shaping the investigated cytochrome as a highly suitable model protein for advanced serial crystallography at highly brilliant X-ray free-electron laser sources.
Asunto(s)
Proteínas Bacterianas/química , Citocromos c6/química , Procesamiento Proteico-Postraduccional , Secuencias de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Reactores Biológicos , Cristalografía por Rayos X , Citocromos c6/genética , Citocromos c6/metabolismo , Expresión Génica , Metilación , Modelos Moleculares , Fotosíntesis/fisiología , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Thermosynechococcus/química , Thermosynechococcus/enzimología , Thermosynechococcus/genéticaRESUMEN
Previous studies have demonstrated that the independent stimulation of either electron transport or RuBP regeneration can increase the rate of photosynthetic carbon assimilation and plant biomass. In this paper, we present evidence that a multigene approach to simultaneously manipulate these two processes provides a further stimulation of photosynthesis. We report on the introduction of the cyanobacterial bifunctional enzyme fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase or the overexpression of the plant enzyme sedoheptulose-1,7-bisphosphatase, together with the expression of the red algal protein cytochrome c6, and show that a further increase in biomass accumulation under both glasshouse and field conditions can be achieved. Furthermore, we provide evidence that the stimulation of both electron transport and RuBP regeneration can lead to enhanced intrinsic water-use efficiency under field conditions.
Asunto(s)
Producción de Cultivos/métodos , Nicotiana/metabolismo , Fotosíntesis , Agua/metabolismo , Cianobacterias/enzimología , Cianobacterias/genética , Cianobacterias/metabolismo , Citocromos c6/genética , Citocromos c6/metabolismo , Transporte de Electrón , Fructosa-Bifosfatasa/genética , Fructosa-Bifosfatasa/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Nicotiana/crecimiento & desarrolloRESUMEN
Plastocyanin and cytochrome c6, abundant proteins in photosynthesis, are readouts for cellular copper status in Chlamydomonas and other algae. Their accumulation is controlled by a transcription factor copper response regulator (CRR1). The replacement of copper-containing plastocyanin with heme-containing cytochrome c6 spares copper and permits preferential copper (re)-allocation to cytochrome oxidase. Under copper-replete situations, the quota depends on abundance of various cuproproteins and is tightly regulated, except under zinc-deficiency where acidocalcisomes over-accumulate Cu(I). CRR1 has a transcriptional activation domain, a Zn-dependent DNA binding SBP-domain with a nuclear localization signal, and a C-terminal Cys-rich region that represses the zinc regulon. CRR1 activates >60 genes in Chlamydomonas through GTAC-containing CuREs; transcriptome differences are recapitulated in the proteome. The differentially-expressed genes encode assimilatory copper transporters of the CTR/SLC31 family including a novel soluble molecule, redox enzymes in the tetrapyrrole pathway that promote chlorophyll biosynthesis and photosystem 1 accumulation, and other oxygen-dependent enzymes, which may influence thylakoid membrane lipids, specifically polyunsaturated galactolipids and γ-tocopherol. CRR1 also down-regulates 2 proteins in Chlamydomonas: for plastocyanin, by activation of proteolysis, while for the diiron subunit of the cyclase in chlorophyll biosynthesis, through activation of an upstream promoter that generates a poorly-translated 5' extended transcript containing multiple short ORFs that inhibit translation. The functions of many CRR1-target genes are unknown, and the copper protein inventory in Chlamydomonas includes several whose functions are unexplored. The comprehensive picture of cuproproteins and copper homeostasis in this system is well-suited for reverse genetic analyses of these under-investigated components in copper biology.
Asunto(s)
Chlamydomonas/genética , Cobre/metabolismo , Fotosíntesis/genética , Transcriptoma/genética , Chlamydomonas/metabolismo , Citocromos c6/genética , Dihidrodipicolinato-Reductasa/genética , Complejo IV de Transporte de Electrones/genética , Regulación de la Expresión Génica de las Plantas/genética , Homeostasis/genética , Plastocianina/genéticaRESUMEN
In this study, we investigate the thermodynamic mechanisms by which electron transfer proteins adapt to environmental temperature by directly comparing the redox properties and folding stability of a psychrophilic cytochrome c and a mesophilic homolog. Our model system consists of two cytochrome c6 proteins from diatoms: one adapted specifically to polar environments, the other adapted generally to surface ocean environments. Direct electrochemistry shows that the midpoint potential for the mesophilic homolog is slightly higher at all temperatures measured. Cytochrome c6 from the psychrophilic diatom unfolds with a melting temperature 10.4 °C lower than the homologous mesophilic cytochrome c6. Changes in free energy upon unfolding are identical, within error, for the psychrophilic and mesophilic protein; however, the chemical unfolding transition of the psychrophilic cytochrome c6 is more cooperative than for the mesophilic cytochrome c6. Substituting alanine residues found in the mesophile with serine found in corresponding positions of the psychrophile demonstrates that burial of the polar serine both decreases the thermal stability and decreases the midpoint potential. The mutagenesis data, combined with differences in the m-value of chemical denaturation, suggest that differences in solvent accessibility of the hydrophobic core underlie the adaptation of cytochrome c6 to differing environmental temperature.
Asunto(s)
Alanina/química , Citocromos c6/química , Serina/química , Termodinámica , Adaptación Fisiológica , Alanina/metabolismo , Secuencia de Aminoácidos , Citocromos c6/genética , Citocromos c6/metabolismo , Diatomeas , Transporte de Electrón , Desplegamiento Proteico , Scenedesmus/enzimología , Alineación de Secuencia , Serina/metabolismoRESUMEN
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.
Asunto(s)
Chlamydomonas reinhardtii/efectos de los fármacos , Cobre/deficiencia , Nanopartículas del Metal/toxicidad , Oxidorreductasas/genética , Proteínas de Plantas/genética , Plata/toxicidad , Secuencia de Bases , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Citocromos c6/genética , Ferredoxinas/genética , Fotosíntesis/efectos de los fármacos , Fotosíntesis/genética , Transcriptoma/efectos de los fármacosRESUMEN
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.
Asunto(s)
Citocromos c6/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Clorofila/metabolismo , Citocromos c6/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Oxidación-Reducción , Fotosíntesis/genética , Complejo de Proteína del Fotosistema II/genética , Proteínas de Plantas/genética , Algas Marinas/metabolismo , Tilacoides/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Transfección/métodos , Ulva/metabolismoRESUMEN
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.
Asunto(s)
Citocromos c6/genética , Níquel/metabolismo , Plastocianina/genética , Synechocystis/crecimiento & desarrollo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Citocromos c6/metabolismo , Regulación Bacteriana de la Expresión Génica , Plastocianina/metabolismo , Synechocystis/genética , Synechocystis/metabolismo , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Chlamydomonas/metabolismo , Cobre/metabolismo , Consumo de Oxígeno/fisiología , Fotosíntesis/fisiología , Ceruloplasmina/genética , Ceruloplasmina/metabolismo , Chlamydomonas/genética , Citocromos c6/genética , Citocromos c6/metabolismo , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Plastocianina/genética , Plastocianina/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/genética , Cobre/toxicidad , Contaminantes Ambientales/toxicidad , Regulación Bacteriana de la Expresión Génica , Proteínas de Transporte de Membrana/genética , Synechocystis/efectos de los fármacos , Transcriptoma , Proteínas Bacterianas/metabolismo , Citocromos c6/genética , Citocromos c6/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Proteínas de Transporte de Membrana/metabolismo , Anotación de Secuencia Molecular , Mutación , Operón , Plastocianina/genética , Plastocianina/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Synechocystis/genética , Synechocystis/metabolismoRESUMEN
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.
Asunto(s)
Chlamydomonas reinhardtii/genética , Cloroplastos/genética , Transgenes/genética , Regiones no Traducidas 5'/genética , Biolística/métodos , Cloroplastos/metabolismo , Cobre/farmacología , Citocromos c6/genética , Expresión Génica , Regulación de la Expresión Génica de las Plantas , Nucleotidiltransferasas/genética , Complejo de Proteína del Fotosistema II/genética , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , Proteínas Represoras/genética , Transformación GenéticaRESUMEN
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.
Asunto(s)
Citocromos c6/química , Synechococcus/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Cristalografía por Rayos X , Citocromos c6/genética , Hemo/química , Enlace de Hidrógeno , Modelos Moleculares , Oxidación-Reducción , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Synechococcus/genéticaRESUMEN
The binding and electron transfer between plastocyanin (pc) or cytochrome c(6) (cyt c(6)) and photosystem I (PSI) can be described by hydrophobic as well as electrostatic interactions. The two α helices, l and l' in PsaB and PsaA, respectively, are involved in forming the hydrophobic interaction site at the oxidizing site of PSI. To obtain mechanistic insights into the function of the two negatively charged residues D612 and E613, present in α helix l of PsaB, we exchanged both residues by site-directed mutagenesis with His and transformed a PsaB deficient mutant of Chlamydomonas reinhardtii. Flash-induced absorption spectroscopy revealed that PSI harboring the changes D612H and E613H had a high affinity toward binding of the electron donors and possessed an altered pH dependence of electron transfer with pc and cyt c(6). Despite optimized binding and electron transfer between the altered PSI and its electron donors, the mutant strain PsaB-D612H/E613H exhibited a strong light sensitive growth phenotype, indicating that decelerated turnover between pc/cyt c(6) and PSI with respect to electron transfer is deleterious to the cells.
Asunto(s)
Chlamydomonas reinhardtii/enzimología , Citocromos c6/química , Complejo de Proteína del Fotosistema I/química , Plastocianina/química , Ácido Aspártico/química , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Chlamydomonas reinhardtii/genética , Citocromos c6/genética , Citocromos c6/metabolismo , Transporte de Electrón/fisiología , Ácido Glutámico/química , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , Concentración de Iones de Hidrógeno , Complejo de Proteína del Fotosistema I/genética , Complejo de Proteína del Fotosistema I/metabolismo , Plastocianina/genética , Plastocianina/metabolismo , Unión ProteicaRESUMEN
Most organisms performing oxygenic photosynthesis contain either cytochrome c(6) or plastocyanin, or both, to transfer electrons from cytochrome b(6)-f to photosystem I. Even though plastocyanin has superseded cytochrome c(6) along evolution, plants contain a modified cytochrome c(6), the so called cytochrome c(6A), whose function still remains unknown. In this article, we describe a second cytochrome c(6) (the so called cytochrome c(6)-like protein), which is found in some cyanobacteria but is phylogenetically more related to plant cytochrome c(6A) than to cyanobacterial cytochrome c(6). In this article, we conclude that the cytochrome c(6)-like protein is a putative electron donor to photosystem I, but does play a role different to that of cytochrome c(6) and plastocyanin as it cannot accept electrons from cytochrome f. The existence of this third electron donor to PSI could explain why some cyanobacteria are able to grow photoautotrophically in the absence of both cytochrome c(6) and plastocyanin. In any way, the Cyt c(6)-like protein from Nostoc sp. PCC 7119 would be potentially utilized for the biohydrogen production, using cell-free photosystem I catalytic nanoparticles.
Asunto(s)
Proteínas Bacterianas/metabolismo , Citocromos c6/metabolismo , Nostoc/metabolismo , Complejo de Proteína del Fotosistema I/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Clonación Molecular , Citocromos c6/química , Citocromos c6/genética , Citocromos c6/aislamiento & purificación , ADN Bacteriano/química , Transporte de Electrón , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Luz , Modelos Moleculares , Datos de Secuencia Molecular , Nostoc/genética , Nostoc/fisiología , Oxidación-Reducción , Fotosíntesis/fisiología , Filogenia , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
The c-type cytochromes are metalloproteins with a heme molecule covalently linked to the sulfhydryls of a CXXCH heme-binding site. In plastids, at least six assembly factors are required for heme attachment to the apo-forms of cytochrome f and cytochrome c(6) in the thylakoid lumen. CCS5, controlling plastid cytochrome c assembly, was identified through insertional mutagenesis in the unicellular green alga Chlamydomonas reinhardtii. The complementing gene encodes a protein with similarity to Arabidopsis thaliana HCF164, which is a thylakoid membrane-anchored protein with a lumen-facing thioredoxin-like domain. HCF164 is required for cytochrome b(6)f biogenesis, but its activity and site of action in the assembly process has so far remained undeciphered. We show that CCS5 is a component of a trans-thylakoid redox pathway and operates by reducing the CXXCH heme-binding site of apocytochrome c prior to the heme ligation reaction. The proposal is based on the following findings: 1) the ccs5 mutant is rescued by exogenous thiols; 2) CCS5 interacts with apocytochrome f and c(6) in a yeast two-hybrid assay; and 3) recombinant CCS5 is able to reduce a disulfide in the CXXCH heme-binding site of apocytochrome f.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Citocromos c6/metabolismo , Citocromos c/metabolismo , Proteínas Protozoarias/metabolismo , Tiorredoxinas/metabolismo , Tilacoides/metabolismo , Animales , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sitios de Unión , Chlamydomonas reinhardtii/genética , Citocromos c/genética , Citocromos c6/genética , Citocromos f/genética , Citocromos f/metabolismo , Hemo/genética , Hemo/metabolismo , Mutación , Oxidación-Reducción , Proteínas Protozoarias/genética , Tiorredoxinas/genética , Tilacoides/genéticaRESUMEN
The electron shuttle heme protein Cyt-c(6) from the photosynthetic cyanobacterium Nostoc sp. PCC 7119 was immobilized on nanostructured Ag electrodes coated with SAMs that mimic different possible interactions with its natural reaction partner PSI. The structure, redox potential, and electron-transfer dynamics of the SAM-Cyt-c(6) complexes were investigated by TR-SERR spectroelectrochemistry. It is shown that the heterogeneous electron-transfer process is gated both in electrostatic and hydrophobic-hydrophilic complexes. At long tunneling distances, the reaction rate is controlled by the tunneling probability, while at shorter distances or higher driving forces, protein dynamics becomes the rate-limiting event.
Asunto(s)
Biomimética , Citocromos c6/química , Electrones , Nostoc/enzimología , Sitios de Unión , Citocromos c6/genética , Cinética , Modelos Biológicos , Mutación , Espectrometría Raman , Propiedades de SuperficieRESUMEN
The primary sequence of cytochrome c(6) from the brown alga Hizikia fusiformis has been determined by cDNA cloning and the crystal structure has been solved at 1.6 A resolution. The crystal belonged to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 84.58, c = 232.91 A and six molecules per asymmetric unit. The genome code, amino-acid sequence and crystal structure of H. fusiformis cytochrome c(6) were most similar to those of red algal cytochrome c(6). These results support the hypothesis that brown algae acquired their chloroplasts via secondary endosymbiosis involving a red algal endosymbiont and a eukaryote host.
Asunto(s)
Citocromos c6/genética , Citocromos c6/aislamiento & purificación , Phaeophyceae/enzimología , Secuencia de Bases , Clonación Molecular , Cristalografía por Rayos X , Citocromos c6/química , Cartilla de ADN , Conformación ProteicaRESUMEN
Photosynthetic plants convert light energy into ATP and NADPH in photosynthetic electron transfer and photophosphorylation, and synthesize mainly carbohydrates in the Calvin-Benson cycle. Here we report the enhancement of photosynthesis and growth of plants by introducing the gene of an algal cytochrome c6, which has been evolutionarily eliminated from higher plant chloroplasts, into the model plant Arabidopsis thaliana. At 60 d after planting, the plant height, leaf length and root length of the transformants were 1.3-, 1.1- and 1.3-fold those in the wild-type plants, respectively. At the same time, in the transgenic plants, the amounts of chlorophyll, protein, ATP, NADPH and starch were 1.2-, 1.1-, 1.9-, 1.4- and 1.2-fold those in the wild-type plants, respectively. The CO2 assimilation capacity of the transgenic plants was 1.3-fold that of the wild type. Moreover, in transgenic Arabidopsis expressing algal cytochrome c6, the 1-qP, which reflects the reduced state of the plastoquinone pool, is 30% decreased compared with the wild type. These results show that the electron transfer of photosynthesis of Arabidopsis would be accelerated by the expression of algal cytochrome c6. Our results demonstrate that the growth and photosynthesis of Arabidopsis plants could be enhanced by the expression of the algal cytochrome c6 gene.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Citocromos c6/genética , Citocromos c6/metabolismo , Eucariontes/enzimología , Eucariontes/genética , Fotosíntesis/fisiología , Transgenes/genética , Arabidopsis/metabolismo , Transporte de Electrón , Expresión Génica , Plantas Modificadas Genéticamente , Factores de TiempoRESUMEN
The reduction of the photo-oxidized special chlorophyll pair P700 of photosystem I (PSI) in the photosynthetic electron transport chain of eukaryotic organisms is facilitated by the soluble copper-containing protein plastocyanin (pc). In the absence of copper, pc is functionally replaced by the heme-containing protein cytochrome c6 (cyt c6) in the green alga Chlamydomonas reinhardtii. Binding and electron transfer between both donors and PSI follows a two-step mechanism that depends on electrostatic and hydrophobic recognition between the partners. Although the electrostatic and hydrophobic recognition sites on pc and PSI are well known, the precise electrostatic recognition site on cyt c6 is unknown. To specify the interaction sites on a molecular level, we cross-linked cyt c6 and PSI using a zero-length cross-linker and obtained a cross-linked complex competent in fast and efficient electron transfer. As shown previously, cyt c6 cross-links specifically with the PsaF subunit of PSI. Mass spectrometric analysis of tryptic peptides from the cross-linked product revealed specific interaction sites between residues Lys27 of PsaF and Glu69 of cyt c6 and between Lys23 of PsaF and Glu69/Glu70 of cyt c6. Using these new data, we present a molecular model of the intermolecular electron transfer complex between eukaryotic cyt c6 and PSI.