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1.
Proc Biol Sci ; 276(1660): 1347-54, 2009 Apr 07.
Article in English | MEDLINE | ID: mdl-19141418

ABSTRACT

It has been proposed that plants are capable of producing methane by a novel and unidentified biochemical pathway. Emission of methane with an apparently biological origin was recorded from both whole plants and detached leaves. This was the first report of methanogenesis in an aerobic setting, and was estimated to account for 10-45 per cent of the global methane source. Here, we show that plants do not contain a known biochemical pathway to synthesize methane. However, under high UV stress conditions, there may be spontaneous breakdown of plant material, which releases methane. In addition, plants take up and transpire water containing dissolved methane, leading to the observation that methane is released. Together with a new analysis of global methane levels from satellite retrievals, we conclude that plants are not a major source of the global methane production.


Subject(s)
Chlamydomonas/metabolism , Methane/metabolism , Phylogeny , Plants/genetics , Plants/metabolism , Animals
2.
Biochem Soc Trans ; 33(Pt 4): 832-5, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16042609

ABSTRACT

The respiratory chain of cyanobacteria appears to be branched rather than linear; furthermore, respiratory and photosynthetic electron-transfer chains co-exist in the thylakoid membrane and even share components. This review will focus on the three types of terminal respiratory oxidases identified so far on a genetic level in cyanobacteria: aa3-type cytochrome c oxidase, cytochrome bd-quinol oxidase and the alternative respiratory terminal oxidase. We summarize here their genetic, biochemical and biophysical characterization to date and discuss their interactions with electron donors as well as their physiological roles.


Subject(s)
Cyanobacteria/enzymology , Electron Transport Complex IV/metabolism , Bacterial Proteins/metabolism , Cell Membrane/drug effects , Cell Membrane/metabolism , Cytochrome b Group , Cytochromes/metabolism , Electron Transport Chain Complex Proteins/metabolism , Electron Transport Complex IV/genetics , Energy Metabolism , Escherichia coli Proteins/metabolism , Kinetics , Operon , Oxidoreductases/metabolism , Oxygen Consumption
3.
Eur J Biochem ; 267(12): 3461-8, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10848961

ABSTRACT

The role of electrostatic interactions in determining the rate of electron transfer between cytochrome f and plastocyanin has been examined in vitro with mutants of turnip cytochrome f and mutants of pea and spinach plastocyanins. Mutation of lysine residues Lys58, Lys65 and Lys187 of cytochrome f to neutral or acidic residues resulted in decreased binding constants and decreased rates of electron transfer to wild-type pea plastocyanin. Interaction of the cytochrome f mutant K187E with the pea plastocyanin mutant D51K gave a further decrease in electron transfer rate, indicating that a complementary charge pair at these positions could not compensate for the decreased overall charge on the proteins. Similar results were obtained with the interaction of the cytochrome f mutant K187E with single, double and triple mutants of residues in the acidic patches of spinach plastocyanin. These results suggest that the lysine residues of the basic patch on cytochrome f are predominantly involved in long-range electrostatic interactions with plastocyanin. However, analysis of the data using thermodynamic cycles provided evidence for the interaction of Lys187 of cytochrome f with Asp51, Asp42 and Glu43 of plastocyanin in the complex, in agreement with a structural model of a cytochrome f-plastocyanin complex determined by NMR.


Subject(s)
Cytochromes/chemistry , Cytochromes/metabolism , Plastocyanin/chemistry , Plastocyanin/metabolism , Aspartic Acid , Brassica/enzymology , Circular Dichroism , Cytochromes/genetics , Cytochromes f , Lysine , Magnetic Resonance Spectroscopy , Mutagenesis, Site-Directed , Oxidation-Reduction , Protein Conformation , Spectrophotometry, Ultraviolet , Static Electricity , Thermodynamics
4.
Biochemistry ; 39(20): 5971-6, 2000 May 23.
Article in English | MEDLINE | ID: mdl-10821668

ABSTRACT

Cytochrome f of oxygenic photosynthesis has an unprecedented structure, including the N-terminus being a heme ligand. The adjacent N-terminal heme-shielding domain is enriched in aromatic amino acids. The atomic structures of the chloroplast and cyanobacterial cytochromes f were compared to explain spectral and redox differences between them. The conserved aromatic side chain in the N-terminal heme-shielding peptide at position 4, Phe and Tyr in plants and algae, respectively, and Trp in cyanobacteria, is in contact with the heme. Mutagenesis of cytochrome f from the eukaryotic green alga Chlamydomonas reinhardtii showed that a Phe4 --> Trp substitution in the N-terminal domain was unique in causing a red shift of 1 and 2 nm in the cytochrome Soret (gamma) and Q (alpha) visible absorption bands, respectively. The resulting alpha band peak at 556 nm is characteristic of the cyanobacterial cytochrome. Conversely, a Trp4 --> Phe mutation in the expressed cytochrome from the cyanobacterium Phormidium laminosum caused a blue shift to the 554 nm alpha band peak diagnostic of the chloroplast cytochrome. Residue 4 was found to be the sole determinant of this 60 cm(-)(1) spectral shift, and of approximately one-half of the 70 mV redox potential difference between cytochrome f of P. laminosum and C. reinhardtii (E(m7) = 297 and 370 mV, respectively). The proximity of Trp-4 to the heme implies that the spectral and redox potential shifts arise through differential interaction of its sigma- or pi-electrostatic potential with the heme ring and of the pi-potential with the heme Fe orbitals, respectively. The dependence of the visible spectrum and redox potential of cytochrome f on the identity of aromatic residue 4 provides an example of the use of the relatively sharp cytochrome spectrum as a "spectral fingerprint", and of the novel structural connection between the heme and a single nonliganding residue.


Subject(s)
Cytochromes/chemistry , Heme/chemistry , Photosynthesis , Tryptophan/chemistry , Amino Acid Sequence , Amino Acid Substitution/genetics , Animals , Chlamydomonas reinhardtii/enzymology , Chlamydomonas reinhardtii/genetics , Chloroplasts/enzymology , Chloroplasts/genetics , Cyanobacteria/enzymology , Cyanobacteria/genetics , Cytochromes/genetics , Cytochromes/metabolism , Cytochromes f , Heme/genetics , Heme/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Oxidation-Reduction , Phenylalanine/genetics , Photosynthesis/genetics , Static Electricity , Tryptophan/genetics , Tryptophan/metabolism , Tyrosine/genetics
5.
Biochemistry ; 38(30): 9590-9, 1999 Jul 27.
Article in English | MEDLINE | ID: mdl-10423236

ABSTRACT

Cytochrome f from the photosynthetic cytochrome b(6)f complex is unique among c-type cytochromes in its fold and heme ligation. The 1. 9-A crystal structure of the functional, extrinsic portion of cytochrome f from the thermophilic cyanobacterium Phormidium laminosum demonstrates that an unusual buried chain of five water molecules is remarkably conserved throughout the biological range of cytochrome f from cyanobacteria to plants [Martinez et al. (1994) Structure 2, 95-105]. Structure and sequence conservation of the cytochrome f extrinsic portion is concentrated at the heme, in the buried water chain, and in the vicinity of the transmembrane helix anchor. The electrostatic surface potential is variable, so that the surface of P. laminosum cytochrome f is much more acidic than that from turnip. Cytochrome f is unrelated to cytochrome c(1), its functional analogue in the mitochondrial respiratory cytochrome bc(1) complex, although other components of the b(6)f and bc(1) complexes are homologous. Identical function of the two complexes is inferred for events taking place at sites of strong sequence conservation. Conserved sites throughout the entire cytochrome b(6)f/bc(1) family include the cluster-binding domain of the Rieske protein and the heme b and quinone-binding sites on the electrochemically positive side of the membrane within the b cytochrome, but not the putative quinone-binding site on the electrochemically negative side.


Subject(s)
Cyanobacteria/enzymology , Cytochromes/chemistry , Bacterial Proteins/chemistry , Brassica/enzymology , Computer Simulation , Conserved Sequence , Crystallography, X-Ray , Cytochrome b Group/chemistry , Cytochrome b6f Complex , Cytochromes f , Electron Transport Complex III/chemistry , Models, Molecular , Plant Proteins/chemistry , Protein Structure, Tertiary , Solubility , Static Electricity , Structure-Activity Relationship , Surface Properties , Water/chemistry
6.
Gene ; 234(2): 275-83, 1999 Jul 08.
Article in English | MEDLINE | ID: mdl-10395900

ABSTRACT

The gene for plastocyanin from the cyanobacterium Phormidium laminosum was successfully expressed in Escherichia coli. Expression of the gene for cytochrome f resulted in the production of holocytochrome f in the periplasmic space of E. coli, but the yield was low. Expression in Paracoccus denitrificans yielded no holoprotein. When the region encoding the cytochrome f leader sequence was replaced with more typical bacterial leader sequences (those from the P. laminosum plastocyanin gene and the Paracoccus versutus cytochrome c-550 gene), much higher yields were consistently obtained in both species. Overexpressed proteins were compared to those isolated from P. laminosum and found to be identical in mass, isoelectric point, redox midpoint potential and (for plastocyanin) 1H-NMR spectrum.


Subject(s)
Cytochromes/genetics , Gram-Negative Bacteria/genetics , Plastocyanin/genetics , Protein Sorting Signals/physiology , Algal Proteins/genetics , Amino Acid Sequence , Bacterial Proteins/genetics , Cyanobacteria/genetics , Cytochrome c Group/genetics , Cytochromes f , Escherichia coli/genetics , Gene Expression Regulation , Paracoccus denitrificans/genetics , Plasmids/genetics , Protein Sorting Signals/genetics
7.
Acta Crystallogr D Biol Crystallogr ; 55(Pt 2): 414-21, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10089349

ABSTRACT

The crystal structure of the 'blue' copper protein plastocyanin from the cyanobacterium Phormidium laminosum has been solved and refined using 2.8 A X--ray data. P. laminosum plastocyanin crystallizes in space group P43212 with unit-cell dimensions a = 86.57, c = 91.47 A and with three protein molecules per asymmetric unit. The final residual R is 19.9%. The structure was solved using molecular replacement with a search model based on the crystal structure of a close homologue, Anabaena variabilis plastocyanin (66% sequence identity). The molecule of P. laminosum plastocyanin has 105 amino-acid residues. The single Cu atom is coordinated by the same residues - two histidines, a cysteine and a methionine - as in other plastocyanins. In the crystal structure, the three molecules of the asymmetric unit are related by a non-crystallographic threefold axis. A Zn atom lies between each pair of neighbouring molecules in this ensemble, being coordinated by a surface histidine residue of one molecule and by two aspartates of the other.


Subject(s)
Cyanobacteria/chemistry , Plastocyanin/chemistry , Amino Acid Sequence , Crystallography, X-Ray , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Protein Conformation , Sequence Homology, Amino Acid , Zinc/chemistry
8.
Eur J Biochem ; 255(1): 309-16, 1998 Jul 01.
Article in English | MEDLINE | ID: mdl-9692933

ABSTRACT

PsaD is a small, extrinsic polypeptide located on the stromal side (cytoplasmic side in cyanobacteria) of the photosystem I reaction centre complex. The gene from the cyanobacterium Nostoc sp. PCC 8009 was expressed in Escherichia coli and the structure of the recovered protein in solution investigated. Size-exclusion chromatography, dynamic light scattering and measurement of 15N transverse relaxation times showed that the protein is a stable dimer in solution, whereas in the reaction centre complex it is a monomer. NMR experiments showed that the dimer is symmetrical and that there are at least two domains, one structured and the remainder unstructured. The structured domain contains a small amount of beta-sheet. Three-dimensional heteronuclear NMR spectra of [13C, 15N]PsaD showed that the structured domain is associated with the central part of the sequence while the N- and C-terminal regions are mobile. Evidence was obtained for a shift in equilibrium between two slightly different conformational states at about pH 6, and the protein was shown to bind to PsaE preferentially at neutral pH. Addition of trifluoroethanol was shown to induce the formation of a small amount of alpha-helix, and the form present in 30% trifluoroethanol appears to be more closely related to the in situ structure, which has been reported to contain one short helix in crystals [Schubert, W.-D., Klukas, O., Krauss, N., Saenger, W., Fromme, P. & Witt, H. T. (1997) J. Mol. Biol. 272, 741-769]. The significance of these findings for the assembly of the complex is discussed.


Subject(s)
Photosynthetic Reaction Center Complex Proteins/chemistry , Photosystem I Protein Complex , Plant Proteins/chemistry , Amino Acid Sequence , Circular Dichroism , Cyanobacteria , Dimerization , Light , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Photosynthetic Reaction Center Complex Proteins/genetics , Photosynthetic Reaction Center Complex Proteins/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Binding , Protein Conformation , Protein Structure, Secondary , Recombinant Proteins/chemistry , Scattering, Radiation , Solutions
9.
Structure ; 6(3): 323-35, 1998 Mar 15.
Article in English | MEDLINE | ID: mdl-9551554

ABSTRACT

BACKGROUND: The reduction of plastocyanin by cytochrome f is part of the chain of photosynthetic electron transfer reactions that links photosystems II and I. The reaction is rapid and is influenced by charged residues on both proteins. Previously determined structures show that the plastocyanin copper and cytochrome f haem redox centres are some distance apart from the relevant charged sidechains, and until now it was unclear how a transient electrostatic complex can be formed that brings the redox centres sufficiently close for a rapid reaction. RESULTS: A new approach was used to determine the structure of the transient complex between cytochrome f and plastocyanin. Diamagnetic chemical shift changes and intermolecular pseudocontact shifts in the NMR spectrum of plastocyanin were used as input in restrained rigid-body molecular dynamics calculations. An ensemble of ten structures was obtained, in which the root mean square deviation of the plastocyanin position relative to cytochrome f is 1.0 A. Electrostatic interaction is maintained at the same time as the hydrophobic side of plastocyanin makes close contact with the haem area, thus providing a short electron transfer pathway (Fe-Cu distance 10.9 A) via residues Tyr1 or Phe4 (cytochrome f) and the copper ligand His87 (plastocyanin). CONCLUSIONS: The combined use of diamagnetic and paramagnetic chemical shift changes makes it possible to obtain detailed information about the structure of a transient complex of redox proteins. The structure suggests that the electrostatic interactions 'guide' the partners into a position that is optimal for electron transfer, and which may be stabilised by short-range interactions.


Subject(s)
Cytochromes/chemistry , Cytochromes/metabolism , Plastocyanin/chemistry , Plastocyanin/metabolism , Binding Sites , Cytochromes f , Electron Transport , Magnetic Resonance Spectroscopy/methods , Models, Molecular , Protein Binding , Protein Conformation
10.
Biochemistry ; 36(21): 6326-35, 1997 May 27.
Article in English | MEDLINE | ID: mdl-9174347

ABSTRACT

The complexes of horse ferrous and ferric cytochrome c with Cd-substituted pea plastocyanin have been characterized by nuclear magnetic resonance, in order to determine the binding sites and to study the effects of complex formation. Reproducible, small chemical shift changes (0.005-0.05 ppm) were observed for protons in both proteins upon formation of a 1:1 complex. The chemical shift changes depended on the ratio of free to bound protein, with a binding constant of 1.0 +/- 0.5 x 10(5) M(-1), indicating that they were caused by complex formation and that free and bound proteins were in fast exchange. Two-dimensional spectra of the complex of ferrocytochrome c and plastocyanin were screened systematically for chemical shift changes. For about 760 protons, or 70% of the assigned protons in the two proteins, the chemical shift in the complex could be established. In plastocyanin and cytochrome c 14% and 17% of the protons, respectively, showed a significant chemical shift change. These protons form two groups. The first consists of a limited number of surface-exposed side-chain protons. These map on the so-called east side of plastocyanin and the front side of cytochrome c. This group of chemical shift changes is interpreted as representing direct effects of binding, and the respective surfaces thus represent the binding sites. The second group includes backbone amide protons and a few aliphatic and aromatic protons in the hydrophobic core of each protein. The chemical shift changes of this group are interpreted as secondary, i.e., caused by very small structural changes which are transmitted deep into the core of the protein. Ferric cytochrome c caused the same chemical shift effects in plastocyanin as the ferrous form; no intermolecular paramagnetic effects were observed. The small size of the chemical shifts and the absence of intermolecular paramagnetic shifts and NOEs suggest that the complex consists of a dynamic ensemble of structures which are in fast exchange, rather than a single static complex. This study shows that small, reproducible chemical shifts can be used effectively to characterize protein complexes in detail.


Subject(s)
Cytochrome c Group/chemistry , Plastocyanin/chemistry , Binding Sites , Cadmium/chemistry , Copper/chemistry , Electron Transport , Ferric Compounds/chemistry , Ferrous Compounds/chemistry , Magnetic Resonance Spectroscopy/methods , Oxidation-Reduction , Potentiometry
11.
Eur J Biochem ; 242(1): 132-47, 1996 Nov 15.
Article in English | MEDLINE | ID: mdl-8954163

ABSTRACT

To compare cadmium-substituted plastocyanin with copper plastocyanin, the 1H-NMR spectra of CuI-, CuII- and Cd-plastocyanin from pea have been analyzed. Full assignments of the spectra of CuI- and Cd-plastocyanin indicate chemical shift differences up to 1 ppm. The affected protons are located in the four loops that surround the Cu site. The largest differences were found for protons in the hydrogen bond network which stabilizes this part of the protein. This suggests that the chemical shift differences are caused by very small but extensive structural changes in the network upon replacement of CuI by Cd. For CuII-plastocyanin the resonances of 72% of the protons observed in the CuI form have been identified. Protons within approximately 0.9 nm of the CuII were not observed due to fast paramagnetic relaxation. The protons between 0.9-1.7 nm from the CuII showed chemical shift differences up to 0.4 ppm compared to both CuI- and Cd-plastocyanin. These differences can be predicted assuming that they represent pseudocontact shifts. When corrected for the pseudocontact shift contribution, the CuII-plastocyanin chemical shifts were nearly all identical within error to those of the Cd form, but not of the CuI-plastocyanin, indicating that the CuII-plastocyanin structure, in as far as it can be observed, resembles Cd-rather than CuI-plastocyanin. In a single stretch of residues (64-69) chemical shift differences remained between all three forms after correction. The fact that pseudocontact shifts were observed for protons which were not broadened may be attributable to the weaker distance dependence of the pseudocontact shift effect compared to paramagnetic relaxation. This results in two shells around the Cu atom, an inner paramagnetic shell (0-0.9 nm), in which protons are not observed due to broadening, and an outer paramagnetic shell (0.9-1.7 nm), in which protons can be observed and show pseudocontact shifts. It is concluded that Cd-plastocyanin is a suitable redox-inactive substitute for Cu-plastocyanin.


Subject(s)
Cadmium , Copper , Plastocyanin/chemistry , Amino Acid Sequence , Electron Spin Resonance Spectroscopy , Hydrogen Bonding , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Pisum sativum
12.
Biochim Biophys Acta ; 1276(3): 246-52, 1996 Sep 30.
Article in English | MEDLINE | ID: mdl-8856106

ABSTRACT

Part of the petCA operon was cloned and the sequence of the cytochrome f gene from the moderately thermophilic cyanobacterium Phormidium laminosum determined. A partial sequence of the petC gene encoding the Rieske iron-sulphur protein was also obtained. The cytochrome f gene encodes a mature protein of 385 residues and a leader sequence of 45 residues. The mature protein contains several acidic or neutral residues corresponding to basic residues in the turnip protein. Some of the latter are thought to be important for the interaction with plastocyanin via its "eastern' face. Many of the corresponding residues on the eastern face of P. laminosum plastocyanin are either basic or neutral instead of acidic. These comparisons suggested that the local charges on P. laminosum cytochrome f that are important for its interaction with the homologous plastocyanin may be negative rather than positive. The importance of acidic groups was confirmed by measuring the rates of reduction of horse heart cytochrome c and P. laminosum and spinach plastocyanins by the cytochrome bf complex isolated from P. laminosum. P. laminosum plastocyanin gave the highest rates, which decreased at high ionic strength, confirming the importance of positive local charges on this protein. When extrapolated to infinite ionic strength the rates observed with the two kinds of plastocyanin were similar, but cytochrome c became unreactive. An optimum was observed in the ionic strength response with P. laminosum plastocyanin.


Subject(s)
Cyanobacteria/genetics , Cytochromes/genetics , Cytochromes/metabolism , Plastocyanin/metabolism , Amino Acid Sequence , Base Sequence , Cyanobacteria/chemistry , Cytochrome b Group , Cytochrome b6f Complex , Cytochromes/chemistry , Cytochromes f , Electron Transport , Gene Library , Genes, Bacterial , Models, Chemical , Molecular Sequence Data , Operon , Osmolar Concentration , Polymerase Chain Reaction , Potentiometry , Sequence Analysis , Sequence Homology, Amino Acid
13.
Photosynth Res ; 50(1): 11-21, 1996 Oct.
Article in English | MEDLINE | ID: mdl-24271818

ABSTRACT

Two mutants of plastocyanin have been constructed by site-directed mutagenesis in spinach and pea to elucidate the binding and electron transfer properties between plastocyanin and spinach Photosystem 1. The conserved, surface-exposed Tyr-83 has been replaced by phenylalanine and leucine in plastocyanin from both species and the proteins have been expressed in Escherichia coli. The reaction mechanism of electron transfer from plastocyanins to photooxidized P700 in Photosystem 1 has been studied by laser-flash absorption spectroscopy. The experimental data were interpreted with a model involving a rate-limiting conformational change, preceding the intracomplex electron transfer. The pea proteins show an overall facilitated reaction with spinach Photosystem 1, compared to spinach plastocyanins. The changes are small but significant, indicating a more efficient electron transfer within the transient complex. In addition, for the spinach leucine mutant, the equilibrium within the plastocyanin-Photosystem 1 complex is more displaced towards the active conformation than for the corresponding wild-type. Absorption spectra, EPR and reduction potentials for the mutants are similar to those of the corresponding wild-type, although small shifts are observed in the spectra of the Tyr83Leu proteins. Based on these results, it is suggested that Photosystem 1 from spinach is capable of using both pea and spinach plastocyanin as an efficient electron donor and that the former even can stimulate the Photosystem 1 reduction. The origin of the stimulation is discussed in terms of differences in surface-exposed residues. Since the effects of the mutations are small, it can be concluded that electron transfer to Photosystem 1 does not occur via Tyr-83.

14.
Plant Mol Biol ; 27(1): 179-90, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7865788

ABSTRACT

Plastocyanin from the thermophilic cyanobacterium Phormidium laminosum has been purified, a partial amino acid sequence obtained and the gene cloned and sequenced. The derived amino acid sequence indicates that the plastocyanin protein is initially synthesized with an N-terminal leader sequence of 34 amino acids to direct it across the thylakoid membrane. The leader sequence consists of a positively charged N-terminal region, a hydrophobic region and a cleavage site, which are characteristic both of higher-plant chloroplast thylakoid transfer domains and of bacterial leader peptides. The petE gene and flanking regions have been cloned in Escherichia coli, and the plastocyanin protein is expressed and directed to the periplasmic space, with concomitant processing to the mature form. Targeting to the periplasm and processing of the plastocyanin protein in E. coli appears to be dependent on components of the Sec apparatus, since the unprocessed precursor accumulates in the cytoplasm of a secA mutant. Expression of plastocyanin in E. coli is copper-inducible and apparently controlled at the level of transcription, leading to the conclusion that copper-regulated promoters exist in the regions flanking the gene and are recognized in a heterologous system. Possible implications for gene expression and protein targeting in the cyanobacterium are discussed.


Subject(s)
Copper/pharmacology , Cyanobacteria/genetics , Escherichia coli Proteins , Gene Expression Regulation, Bacterial/drug effects , Genes, Bacterial/genetics , Membrane Transport Proteins , Plastocyanin/genetics , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , Cloning, Molecular , Escherichia coli/genetics , Molecular Sequence Data , Plastocyanin/biosynthesis , Recombinant Proteins/biosynthesis , SEC Translocation Channels , SecA Proteins , Sequence Analysis , Sequence Homology, Amino Acid
15.
Photosynth Res ; 37(2): 147-58, 1993 Aug.
Article in English | MEDLINE | ID: mdl-24317711

ABSTRACT

Photosystem II cyclic electron transport was investigated at low pH in spinach thylakoids and PS II preparations from the cyanobacteriumPhormidium laminosum. Variable fluorescence (Fv) quenching at a very low light intensity was examined as an indicator of cyclic electron flow. A progressive quenching of Fv was observed as the pH was lowered; however, this was shown to be mainly due to an inhibition of oxygen evolution. Cyclic electron flow in the uninhibited centres was estimated to occur at a rate comparable to or smaller than 1 µ mole O2 mg Chl(-1) h(-1) in the pH range 5.0 to 7.8.The quantum yeeld of oxygen production is known to decrease at low pH and has been taken to indicate cyclic electron flow (Crofts and Horton (1991) Biochim Biophys Acta 1058: 187-193). However, a direct all-or-none inhibition of oxygen production at low pH has also been reported (Meyer et al. (1989) Biochim Biophys Acta 974: 36-43). We have analysed the effects of light intensity on the rates of oxygen evolution in order to calculate ΦU, the quantum yield of open and uninhibited centres. ΦU was found to be constant over a broad pH range, and by using ferricyanide and phenyl-p-benzoquinone as electron acceptors the maximum possible rate of cyclic electron transport was equivalent to no more than 1 µmole O2 mg Chl(-1) h(-1). The rate was no greater when the acceptor was adjusted to provide the most favourable conditions for cyclic flow.

16.
Biochim Biophys Acta ; 1102(1): 85-90, 1992 Aug 28.
Article in English | MEDLINE | ID: mdl-1324731

ABSTRACT

The reduction of plastocyanin by cytochromes c and f has been investigated with mutants of spinach plastocyanin in which individual, highly conserved surface residues have been modified. These include Leu-12 and Phe-35 in the 'northern' hydrophobic patch and Tyr-83 and Asp-42 in the 'eastern' acidic patch. The differences observed all involved binding rather than the intrinsic rates of electron transfer. The Glu-12 and Ala-12 mutants showed small but significant decreases in binding constant with cytochrome c, even though the cytochrome is not expected to make contact with the northern face of plastocyanin. These results, and small changes in the EPR parameters, suggested that these mutations cause small conformational changes in surface residues on the eastern face of plastocyanin, transmitted through the copper centre. In the case of cytochrome f, the Glu-12 and Ala-12 mutants also bound less strongly, but Leu12Asn showed a marked increase in binding constant, suggesting that cytochrome f can hydrogen bond directly to Asn-12 in the reaction complex. A surprising result was that the kinetics of reduction of Asp42Asn were not significantly different from wild type, despite the loss of a negative charge.


Subject(s)
Cytochrome c Group/metabolism , Cytochromes/metabolism , Plastocyanin/metabolism , Amino Acid Sequence , Binding Sites , Cytochromes f , Electron Spin Resonance Spectroscopy , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Oxidation-Reduction , Plastocyanin/genetics
17.
Mol Microbiol ; 6(13): 1821-7, 1992 Jul.
Article in English | MEDLINE | ID: mdl-1630319

ABSTRACT

Cytoplasmic and thylakoid membranes have been purified from the cyanobacteria Anacystis nidulans R2 and Phormidium laminosum by sucrose density gradient centrifugation. Probing of Western blots of proteins from these purified membrane fractions with antibodies directed against the 33 kDa polypeptide of Photosystem II from pea indicates that this protein is present in both the thylakoid and cytoplasmic membranes, rather than just the thylakoid membranes. This has been confirmed by immunogold labelling of cells. Oxygen evolution assays have been used to show that the 33 kDa polypeptide is not assembled into a functional Photosystem II complex in the cytoplasmic membranes. This may be due to the absence of other Photosystem II components.


Subject(s)
Bacterial Proteins , Cell Membrane/chemistry , Cyanobacteria/chemistry , Hexosyltransferases , Membrane Proteins/analysis , Peptidyl Transferases , Photosynthetic Reaction Center Complex Proteins/analysis , Blotting, Western , Carotenoids/analysis , Carrier Proteins/analysis , Cell Fractionation , Chlorophyll/analysis , Immunohistochemistry , Intracellular Membranes/chemistry , Membrane Proteins/metabolism , Muramoylpentapeptide Carboxypeptidase/analysis , Oxygen/metabolism , Penicillin-Binding Proteins , Photosynthetic Reaction Center Complex Proteins/metabolism , Pigments, Biological/analysis
18.
Photosynth Res ; 32(2): 109-20, 1992 May.
Article in English | MEDLINE | ID: mdl-24408281

ABSTRACT

There is presently a debate concerning the number of phases in fluorescence induction and on the identification of the several possible heterogeneities in PS II centres. However, the usual methods of analysis present numerical problems, including a lack of 'robustness' (robustness being defined as the ability to give the correct answer in the presence of distortions or artefacts). We present here the adaptation of the method of moments, which was developed for robustness, to the analysis of fluorescence induction. We were thus able to identify three phases in the fluorescence induction in the presence of DCMU. The slowest phase was attributed to the centres inactive in plastoquinone reduction by using duroquinone as electron acceptor. In order to compare fluorescence with and without DCMU, we introduced the 'rate of photochemistry', defined as the product of the area times the rate constant of an exponential. This quantity is invariant for a given centre no matter what the size of the electron acceptor pool is. The two fastest phases in the presence of DCMU were attributed to active centres because their rate of photochemistry was the same as that of the plastoquinone-reducing phases in the absence of DCMU. Because their reduction of plastoquinone showed different kinetics, these two types of active centres were either separated by more than 250 nm or were associated with discrete plastoquinone pools having restricted diffusion domains.

19.
Photosynth Res ; 34(3): 409-18, 1992 Dec.
Article in English | MEDLINE | ID: mdl-24408836

ABSTRACT

Absorbance changes of ferredoxin measured at 463 nm in isolated thylakoids were shown to arise from the activity of the enzyme ferredoxin-plastoquinone reductase (FQR) in cyclic electron transport. Under anaerobic conditions in the presence of DCMU and an appropriate concentration of reduced ferredoxin, a light-induced absorbance decrease due to further reduction of Fd was assigned to the oxidation of the other components in the cyclic pathway, primarily plastoquinone. When the light was turned off, Fd was reoxidised and this gave a direct quantitative measurement of the rate of cyclic electron transport due to the activity of FQR. This activity was sensitive to the classical inhibitor of cyclic electron transport, antimycin, and also to J820 and DBMIB. Antimycin had no effect on Fd reduction although this was inhibited by stigmatellin. This provides further evidence that there is a quinone reduction site outside the cytochrome bf complex. The effect of inhibitors of ferredoxin-NADP(+) reductase and experiments involving the modification of ferredoxin suggest that there may be some role for the reductase as a component of FQR. Contrary to expectations, NADPH2 inhibited FQR activity; ATP and ADP had no effect.

20.
EMBO J ; 10(13): 4011-6, 1991 Dec.
Article in English | MEDLINE | ID: mdl-1756713

ABSTRACT

Site-directed mutants of the pea plastocyanin gene in which the codon for the surface-exposed Tyr83 has been changed to codons for Phe83 and Leu83 have been expressed in transgenic tobacco plants. The mutant proteins have been purified to homogeneity and their conformations shown not to differ significantly from the wild-type plastocyanin by 1H-NMR and CD. Overall rate constants for electron transfer (k2) from cytochrome f to plastocyanin have been measured by stopped-flow spectrophotometry and rate constants for binding (ka) and association constants (KA) have been measured from the enhanced Soret absorption of cytochrome f on binding plastocyanin. These measurements allow the calculation of the intrinsic rate of electron transfer in the binary complex. An 8-fold decrease in the overall rate of electron transfer to the Phe83 mutant is due entirely to a decreased association constant for cytochrome f, whereas the 40-fold decrease in the overall rate of electron transfer to the Leu83 mutant is due to weaker binding and a lower intrinsic rate of electron transfer. This indicates that Tyr83 is involved in binding to cytochrome f and forms part of the main route of electron transfer.


Subject(s)
Cytochromes/metabolism , Fabaceae/genetics , Plants, Medicinal , Plastocyanin/genetics , Tyrosine/genetics , Base Sequence , Binding Sites , Circular Dichroism , Cytochromes f , Deoxyribonucleotides , Electrons , Fabaceae/metabolism , Kinetics , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Mutagenesis, Site-Directed , Plants, Genetically Modified/genetics , Plasmids , Plastocyanin/metabolism , Restriction Mapping , Spectrophotometry, Ultraviolet
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