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Bioelectrochemistry (Amsterdam,... Nov 2009Many fleeting macromolecular interactions, like those being involved in electron transport, are essential in biology. However, little is known about the behaviour of the...
Many fleeting macromolecular interactions, like those being involved in electron transport, are essential in biology. However, little is known about the behaviour of the partners and their dynamics within their short-lived complex. To tackle such issue, we have performed molecular dynamics simulations on an electron transfer complex formed by plastocyanin and cytochrome f from the cyanobacterium Phormidium laminosum. Besides simulations of the isolated partners, two independent trajectories of the complex were calculated, starting from the two different conformations in the NMR ensemble. The first one leads to a more stable ensemble with a shorter distance between the metal sites of the two partners. The second experiences a significant drift of the complex conformation. Analyses of the distinct calculations show that the conformation of cytochrome f is strained upon binding of its partner, and relaxes upon its release. Interestingly, the principal component analysis of the trajectories indicates that plastocyanin displays a concerted motion with the small domain of cytochrome f that can be attributed to electrostatic interactions between the two proteins.
Topics: Cytochromes f; Molecular Dynamics Simulation; Movement; Oscillatoria; Plastocyanin; Protein Binding; Protein Structure, Tertiary; Static Electricity; Time Factors
PubMed: 19616485
DOI: 10.1016/j.bioelechem.2009.06.003 -
Journal of Biochemistry Dec 1983Treatment of isolated spinach thylakoid fragments with Triton X-100 followed by repeated sucrose density gradient centrifugations and Sephacryl S-300 and DEAE-Sephacel...
Treatment of isolated spinach thylakoid fragments with Triton X-100 followed by repeated sucrose density gradient centrifugations and Sephacryl S-300 and DEAE-Sephacel chromatographies yielded a highly purified P700-chlorophyll a protein complex complex which consists of five polypeptides. The protein complex is virtually free of chlorophyll b (Ch1 alpha/Ch1 b greater than 10) with approximately 30 chlorophylls per P700, and contains iron-sulfur centers A, B, and X. At pH values higher than 6, divalent cations, but not monovalent or trivalent cations, efficiently accelerated the electron transfer from reduced spinach plastocyanin to the photooxidized P700 in the P700-chlorophyll alpha protein complex. At pH values lower than 6, the reaction rate drastically increased with decreasing pH with a maximum at about pH 4.3 without cations. Divalent salts as well as monovalent or trivalent salts decreased the P700 reduction rate at low pH, indicating the involvement of electrostatic interaction in those pH regions. The rate of electron transfer from plastocyanin to the photooxidized P700 in the reaction center protein, which consists of only the largest peptide subunit and no iron-sulfur centers, was reduced only 50% at pH 7.0 in the presence of MgCl2 as compared to the case of P700-chlorophyll alpha protein complex. Essentially similar effects of pH and metal ions on this electron transfer reaction were observed as in the case of P700-chlorophyll alpha protein complex. These results strongly suggest that plastocyanin donates electrons directly to the largest peptide of P700-chlorophyll alpha protein complex and the observed effects of pH and cations are mainly due to the interaction between the largest peptide of P700-chlorophyll alpha protein complex and plastocyanin. The four small subunits in the protein complex seemed to have only a minor role in the reaction with plastocyanin.
Topics: Cations; Chlorophyll; Chloroplasts; Electron Transport; Hydrogen-Ion Concentration; Kinetics; Light-Harvesting Protein Complexes; Macromolecular Substances; Molecular Weight; Oxidation-Reduction; Photosynthetic Reaction Center Complex Proteins; Photosystem I Protein Complex; Plant Proteins; Plants; Plastocyanin
PubMed: 6368528
DOI: 10.1093/oxfordjournals.jbchem.a134544 -
Journal of Biochemistry Feb 1992The green alga Pediastrum boryanum synthesizes alternatively two photosynthetic electron carrier proteins, plastocyanin and cytochrome c-553, depending on the copper...
The green alga Pediastrum boryanum synthesizes alternatively two photosynthetic electron carrier proteins, plastocyanin and cytochrome c-553, depending on the copper concentration of the medium. We studied the levels at which the syntheses of the two proteins are regulated. Plastocyanin and cytochrome c-553 were purified from P. boryanum NIES-301 cells, having apparent molecular weights of 14,600 and about 12,000, respectively. Western blotting with antisera raised against these proteins showed accumulation of (apo)plastocyanin and (apo)cytochrome c-553 in the cells grown with (2 microM) and without added CuSO4, respectively, but no accumulation of the precursor proteins in both cultures. The translatable mRNAs for the two proteins were examined by in vitro translation with total RNA and wheat germ extract followed by immunoprecipitation and SDS-PAGE. The 21-kDa polypeptide (preapoplastocyanin) was detected with anti-plastocyanin serum in copper-sufficient cells; the 23-kDa polypeptide (preapocytochrome c-553) with anti-cytochrome c-553 serum in copper-deficient cells. The translatable mRNA for preapoplastocyanin appeared in 1 h and (apo)plastocyanin in 2-3 h after the addition of 2 microM CuSO4 to the copper-deficient culture. The translatable mRNA for preapocytochrome c-553 disappeared within 4-5 h, while (apo)cytochrome c-553 disappeared more slowly. It is concluded that the syntheses of plastocyanin and cytochrome c-553 are regulated by copper at the pre-translational (i.e., transcriptional or post-transcriptional) level in P. boryanum NIES-301.
Topics: Blotting, Western; Cells, Cultured; Chlorophyta; Copper; Cytochrome c Group; Electrophoresis; Plastocyanin; Protein Biosynthesis; RNA, Messenger; Transcription, Genetic
PubMed: 1314811
DOI: 10.1093/oxfordjournals.jbchem.a123740 -
Proceedings of the National Academy of... Aug 2011Copper is supplied to plastocyanin for photosynthesis and cytochrome c oxidase for respiration in the thylakoids of Synechocystis PCC 6803 by the membrane-bound P-type...
Copper is supplied to plastocyanin for photosynthesis and cytochrome c oxidase for respiration in the thylakoids of Synechocystis PCC 6803 by the membrane-bound P-type ATPases CtaA and PacS, and the metallochaperone Atx1. We have determined the Cu(I) affinities of all of the soluble proteins and domains in this pathway. The Cu(I) affinities of the trafficking proteins range from 5 × 10(16) to 5 × 10(17) M(-1) at pH 7.0, consistent with values for homologues. Unusually, Atx1 binds Cu(I) significantly tighter than the metal-binding domains (MBDs) of CtaA and PacS (CtaA(N) and PacS(N)), and equilibrium copper exchange constants of approximately 0.2 are obtained for transfer to the MBDs. Dimerization of Atx1 increases the affinity for Cu(I), but the loop 5 His61 residue has little influence. The MBD of the zinc exporter ZiaA (ZiaA(N)) exhibits an almost identical Cu(I) affinity, and Cu(I) exchange with Atx1, as CtaA(N) and PacS(N), and the relative stabilities of the complexes must enable the metallochaperone to distinguish between the MBDs. The binding of potentially competing zinc to the trafficking proteins has been studied. ZiaA(N) has the highest Zn(II) affinity and thermodynamics could be important for zinc removal from the cell. Plastocyanin has a Cu(I) affinity of 2.6 × 10(17) M(-1), 15-fold tighter than that of the Cu(A) site of cytochrome c oxidase, highlighting the need for specific mechanisms to ensure copper delivery to both of these targets. The narrow range of Cu(I) affinities for the cytoplasmic copper proteins in Synechocystis will facilitate relocation when copper is limiting.
Topics: Bacterial Proteins; Biological Transport; Copper; Kinetics; Models, Biological; Oxidation-Reduction; Plastocyanin; Protein Multimerization; Synechocystis; Thermodynamics; Zinc
PubMed: 21778408
DOI: 10.1073/pnas.1101448108 -
Plant Physiology Aug 2012Photosynthetic organisms need copper for cytochrome oxidase and for plastocyanin in the fundamental processes of respiration and photosynthesis. However, excess of free...
Photosynthetic organisms need copper for cytochrome oxidase and for plastocyanin in the fundamental processes of respiration and photosynthesis. However, excess of free copper is detrimental inside the cells and therefore organisms have developed homeostatic mechanisms to tightly regulate its acquisition, sequestration, and efflux. Herein we show that the CopRS two-component system (also known as Hik31-Rre34) is essential for copper resistance in Synechocystis sp. PCC 6803. It regulates expression of a putative heavy-metal efflux-resistance nodulation and division type copper efflux system (encoded by copBAC) as well as its own expression (in the copMRS operon) in response to the presence of copper in the media. Mutants in this two-component system or the efflux system render cells more sensitive to the presence of copper in the media and accumulate more intracellular copper than the wild type. Furthermore, CopS periplasmic domain is able to bind copper, suggesting that CopS could be able to detect copper directly. Both operons (copMRS and copBAC) are also induced by the photosynthetic inhibitor 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone but this induction requires the presence of copper in the media. The reduced response of two mutant strains to copper, one lacking plastocyanin and a second one impaired in copper transport to the thylakoid, due to the absence of the P(I)-type ATPases PacS and CtaA, suggests that CopS can detect intracellular copper. In addition, a tagged version of CopS with a triple HA epitope localizes to both the plasma and the thylakoid membranes, suggesting that CopS could be involved in copper detection in both the periplasm and the thylakoid lumen.
Topics: Adaptation, Physiological; Bacterial Proteins; Biological Transport; Cell Membrane; Copper; Intracellular Space; Models, Biological; Oxidation-Reduction; Periplasm; Plastocyanin; Promoter Regions, Genetic; Protein Binding; Protein Structure, Tertiary; Protein Transport; Signal Transduction; Synechocystis; Thylakoids
PubMed: 22715108
DOI: 10.1104/pp.112.200659 -
Plant Physiology Nov 2001Oxygen electrode and fluorescence studies demonstrate that linear electron transport in the freshwater alga Chlamydomonas reinhardtii can be completely abolished by...
Oxygen electrode and fluorescence studies demonstrate that linear electron transport in the freshwater alga Chlamydomonas reinhardtii can be completely abolished by abrupt hyperosmotic shock. We show that the most likely primary site of inhibition of electron transfer by hyperosmotic shock is a blockage of electron transfer between plastocyanin (PC) or cytochrome c(6) and P(700). The effects on this reaction were reversible upon dilution of the osmolytes and the stability of plastocyanin or photosystem (PS) I was unaffected. Electron micrographs of osmotically shocked cells showed a significant decrease in the thylakoid lumen volume. Comparison of estimated lumenal width with the x-ray structures of plastocyanin and PS I suggest that lumenal space contracts during HOS so as to hinder the movement of docking to PS I of plastocyanin or cytochrome c(6).
Topics: Animals; Carbohydrates; Chlamydomonas reinhardtii; Chlorophyll; Chlorophyll A; Cytochromes; Cytochromes f; Electron Transport; Fluorescence; Light; Light-Harvesting Protein Complexes; Osmotic Pressure; Oxidation-Reduction; Oxygen; Photosynthetic Reaction Center Complex Proteins; Plastocyanin; Salts; Thylakoids
PubMed: 11706196
DOI: No ID Found -
Photosynthesis Research May 2016A newly developed compact measuring system for assessment of transmittance changes in the near-infrared spectral region is described; it allows deconvolution of redox...
A newly developed compact measuring system for assessment of transmittance changes in the near-infrared spectral region is described; it allows deconvolution of redox changes due to ferredoxin (Fd), P700, and plastocyanin (PC) in intact leaves. In addition, it can also simultaneously measure chlorophyll fluorescence. The major opto-electronic components as well as the principles of data acquisition and signal deconvolution are outlined. Four original pulse-modulated dual-wavelength difference signals are measured (785-840 nm, 810-870 nm, 870-970 nm, and 795-970 nm). Deconvolution is based on specific spectral information presented graphically in the form of 'Differential Model Plots' (DMP) of Fd, P700, and PC that are derived empirically from selective changes of these three components under appropriately chosen physiological conditions. Whereas information on maximal changes of Fd is obtained upon illumination after dark-acclimation, maximal changes of P700 and PC can be readily induced by saturating light pulses in the presence of far-red light. Using the information of DMP and maximal changes, the new measuring system enables on-line deconvolution of Fd, P700, and PC. The performance of the new device is demonstrated by some examples of practical applications, including fast measurements of flash relaxation kinetics and of the Fd, P700, and PC changes paralleling the polyphasic fluorescence rise upon application of a 300-ms pulse of saturating light.
Topics: Chlorophyll; Ferredoxins; Fluorescence; Hedera; Kinetics; Light; Oxidation-Reduction; Plant Leaves; Plastocyanin; Spectrophotometry
PubMed: 26837213
DOI: 10.1007/s11120-016-0219-0 -
Frontiers in Plant Science 2018For modeling approaches in systems biology, knowledge of the absolute abundances of cellular proteins is essential. One way to gain this knowledge is the use of...
For modeling approaches in systems biology, knowledge of the absolute abundances of cellular proteins is essential. One way to gain this knowledge is the use of quantification concatamers (QconCATs), which are synthetic proteins consisting of proteotypic peptides derived from the target proteins to be quantified. The QconCAT protein is labeled with a heavy isotope upon expression in and known amounts of the purified protein are spiked into a whole cell protein extract. Upon tryptic digestion, labeled and unlabeled peptides are released from the QconCAT protein and the native proteins, respectively, and both are quantified by LC-MS/MS. The labeled Q-peptides then serve as standards for determining the absolute quantity of the native peptides/proteins. Here, we have applied the QconCAT approach to for the absolute quantification of the major proteins and protein complexes driving photosynthetic light reactions in the thylakoid membranes and carbon fixation in the pyrenoid. We found that with 25.2 attomol/cell the Rubisco large subunit makes up 6.6% of all proteins in a cell and with this exceeds the amount of the small subunit by a factor of 1.56. EPYC1, which links Rubisco to form the pyrenoid, is eight times less abundant than RBCS, and Rubisco activase is 32-times less abundant than RBCS. With 5.2 attomol/cell, photosystem II is the most abundant complex involved in the photosynthetic light reactions, followed by plastocyanin, photosystem I and the cytochrome b/ complex, which range between 2.9 and 3.5 attomol/cell. The least abundant complex is the ATP synthase with 2 attomol/cell. While applying the QconCAT approach, we have been able to identify many potential pitfalls associated with this technique. We analyze and discuss these pitfalls in detail and provide an optimized workflow for future applications of this technique.
PubMed: 30214453
DOI: 10.3389/fpls.2018.01265 -
PloS One 2023In large areas of the ocean, iron concentrations are insufficient to promote phytoplankton growth. Numerous studies have been conducted to characterize the effect of...
In large areas of the ocean, iron concentrations are insufficient to promote phytoplankton growth. Numerous studies have been conducted to characterize the effect of iron on algae and how algae cope with fluctuating iron concentrations. Fertilization experiments in low-iron areas resulted primarily in diatom-dominated algal blooms, leading to laboratory studies on diatoms comparing low- and high-iron conditions. Here, we focus on the short-term temporal response following iron addition to an iron-starved open ocean diatom, Thalassiosira oceanica. We employed the NanoString platform and analyzed a high-resolution time series on 54 transcripts encoding proteins involved in photosynthesis, N-linked glycosylation, iron transport, as well as transcription factors. Nine transcripts were iron-responsive, with an immediate response to the addition of iron. The fastest response observed was the decrease in transcript levels of proteins involved in iron uptake, followed by an increase in transcript levels of iron-containing enzymes and a simultaneous decrease in the transcript levels of their iron-free replacement enzymes. The transcription inhibitor actinomycin D was used to understand the underlying mechanisms of the decrease of the iron-responsive transcripts and to determine their half-lives. Here, Mn-superoxide dismutase (Mn-SOD), plastocyanin (PETE), ferredoxin (PETF) and cellular repressor of EA1-stimulated genes (CREGx2) revealed longer than average half-lives. Four iron-responsive transcripts showed statistically significant differences in their decay rates between the iron-recovery samples and the actD treatment. These differences suggest regulatory mechanisms influencing gene transcription and mRNA stability. Overall, our study contributes towards a detailed understanding of diatom cell biology in the context of iron fertilization response and provides important observations to assess oceanic diatom responses following sudden changes in iron concentrations.
Topics: Diatoms; Phytoplankton; Oceans and Seas; Photosynthesis
PubMed: 36693065
DOI: 10.1371/journal.pone.0280827 -
Microbiology (Reading, England) May 1994In some cyanobacteria and eukaryotic algae, cytochrome c-553 (c-552) and plastocyanin function as alternative electron carriers between the cytochrome b6-f complex and... (Comparative Study)
Comparative Study
In some cyanobacteria and eukaryotic algae, cytochrome c-553 (c-552) and plastocyanin function as alternative electron carriers between the cytochrome b6-f complex and Photosystem I. In these organisms plastocyanin is the electron carrier under copper-replete conditions, and cytochrome c-553 is the electron carrier during copper deprivation. In this paper we report the cloning, sequencing and transcriptional analysis of the genes for cytochrome c-553 and plastocyanin from Anabaena sp. PCC 7120. The gene for cytochrome c-553 encodes a preprotein containing 111 amino acids with a predicted N-terminal transit peptide sequence of 25 amino acids. The gene for plastocyanin encodes a preprotein containing 139 amino acids with a N-terminal transit peptide sequence of 34 amino acids. RNA transcript analyses indicate that the expression of the genes for cytochrome c-553 (petJ) and plastocyanin (petE) are regulated in reciprocal ways in response to copper concentration. In copper-replete conditions, petJ is expressed at very low levels, but is transcribed at high levels under copper deprivation; petE is down-regulated in the absence of copper, but is rapidly up-regulated when copper is added back to the medium.
Topics: Amino Acid Sequence; Anabaena; Base Sequence; Cloning, Molecular; Cytochrome c Group; DNA Probes; Genes, Bacterial; Molecular Sequence Data; Plastocyanin; Protein Sorting Signals; RNA, Messenger; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Transcription, Genetic
PubMed: 8025680
DOI: 10.1099/13500872-140-5-1151