The Z-scheme--down hill all the way.
The Z-scheme is considered in the context of personal recollections of events during the time that it was conceived and an evaluation of its enduring importance.
Crassulaceae↗
SEARCH · PubMed Health
Results for “Cytochrome b6f Complex”
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Reconstitution of the 2Fe-2S center and g = 1.89 electron paramagnetic resonance signal into overproduced Nostoc sp. PCC 7906 Rieske protein.
The Rieske 2Fe-2S protein is a distinguishing subunit of the photosynthetic electron transport cytochrome b6f complex in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for overproduction in Escherichia coli of fusion, full-length, and truncated forms of the Rieske (PetC) protein from the cyanobacterium Nostoc sp. PCC 7906. A glutathione S-transferase/Rieske fusion protein was used to prepare specific chicken egg-yolk antibodies against the Rieske protein. Expression of the nonfusion petC gene in a T7 RNA polymerase promoter vector produced copious quantities of the full-length Rieske protein predominantly as inclusion bodies. The highly enriched, Rieske protein from inclusion bodies has been denatured in guanidine hydrochloride and refolded and the characteristic 2Fe-2S cluster reconstituted in vitro by incubation with iron and sulfide under reducing conditions. Purification by chromatography on Whatman DE52 cellulose and ultrafiltration through a 30000 molecular weight cutoff membrane yielded pure and predominantly monomeric Rieske protein. Reconstituted Rieske preparations showed intense and highly characteristic gx = 1.74, gy = 1.89, and gz = 2.03 "Rieske-type" electron paramagnetic resonance signals at 15 K. Two methods of reconstitution yielded Rieske preparations in which 20-60% of the protein contained 2Fe-2S clusters as determined by EPR spin quantitation. The reconstituted Rieske protein was soluble and stable at 4 degrees C in buffers containing nonionic detergents and showed a redox midpoint potential of +321 mV at pH 7.0 as determined by optical circular dichroism (CD) spectroscopy. These data demonstrate the in vitro restoration of a Cys and His liganded 2Fe-2S cluster and provide the basis for mutational and structural analysis of a PetC Rieske protein of oxygenic photosynthesis.
Animals↗
Involvement of chloroplasts in the programmed death of plant cells.
The effect of cyanide, an apoptosis inducer, on pea leaf epidermal peels was investigated. Illumination stimulated the CN--induced destruction of guard cells (containing chloroplasts and mitochondria) but not of epidermal cells (containing mitochondria only). The process was prevented by antioxidants (alpha-tocopherol, 2,5-di-tret-butyl-4-hydroxytoluene, and mannitol), by anaerobiosis, by the protein kinase C inhibitor staurosporine, and by cysteine and serine protease inhibitors. Electron acceptors (menadione, p-benzoquinone, diaminodurene, TMPD, DCPIP, and methyl viologen) suppressed CN--induced apoptosis of guard cells, but not epidermal cells. Methyl viologen had no influence on the removal of CN--induced nucleus destruction in guard cells under anaerobic conditions. The light activation of CN--induced apoptosis of guard cells was suppressed by DCMU (an inhibitor of the electron transfer in Photosystem II) and by DNP-INT (an antagonist of plastoquinol at the Qo site of the chloroplast cytochrome b6f complex). It is concluded that apoptosis initiation in guard cells depends on the simultaneous availability of two factors, ROS and reduced quinones of the electron transfer chain. The conditions for manifestation of programmed cell death in guard and epidermal cells of the pea leaf were significantly different.
Apoptosis↗
Sodium dependency of the photosynthetic electron transport in the alkaliphilic cyanobacterium Arthrospira platensis.
Arthrospira (Spirulina) platensis (A. platensis) is a model organism for investigation of adaptation of photosynthetic organisms to extreme environmental conditions: the cell functions in this cyanobacterium are optimized to high pH and high concentration (150-250 mM) of Na+. However, the mechanism of the possible fine-tuning of the photosynthetic functions to these extreme conditions and/or the regulation of the cellular environment to optimize the photosynthetic functions is poorly understood. In this work we investigated the effect of Na-ions on different photosynthetic activities: linear electron transport reactions (measured by means of polarography and spectrophotometry), the activity of photosystem II (PS II) (thermoluminescence and chlorophyll a fluorescence induction), and redox turnover of the cytochrome b6f complex (flash photolysis); and measured the changes of the intracellular pH (9-aminoacridine fluorescence). It was found that sodium deprivation of cells in the dark at pH 10 inhibited, within 40 min, all measured photosynthetic reactions, and led to an alkalinization of the intracellular pH, which rose from the physiological value of about 8.3-9.6. These were partially and totally restored by readdition of Na-ions at 2.5-25 mM and about 200 mM, respectively. The intracellular pH and the photosynthetic functions were also sensitive to monensin, an exogenous Na+/H+ exchanger, which collapses both proton and sodium gradients across the cytoplasmic membrane. These observations explain the strict Na+-dependency of the photosynthetic electron transport at high extracellular pH, provide experimental evidence on the alkalization of the intracellular environment, and support the hypothesized role of an Na+/H+ antiport through the plasma membrane in pH homeostasis (Schlesinger et al. (1996). J. Phycol. 32, 608-613). Further, we show that (i) the specific site of inactivation of the photosynthetic electron transport at alkaline pH is to be found at the water splitting enzyme; (ii) in contrast to earlier reports, the inactivation occurs in the dark and, for short periods, without detectable damage in the photosynthetic apparatus; and (iii) in contrast to high pH, Na+ dependency in the neutral pH range is shown not to originate from PSII, but from the acceptor side of PSI. These data permit us to conclude that the intracellular environment rather than the machinery of the photosynthetic electron transport is adjusted to the extreme conditions of high pH and high Na+ concentration.
Alkalies↗
RNA-binding properties of HCF152, an Arabidopsis PPR protein involved in the processing of chloroplast RNA.
The nonphotosynthetic mutant of Arabidopsis hcf152 is impaired in the processing of the chloroplast polycistronic transcript, psbB-psbT-psbH-petB-petD, resulting in nonproduction of the essential photosynthetic cytochrome b6f complex. The nucleus-encoded HCF152gene was identified to encode a pentatricopeptide repeat (PPR) protein composed primarily of 12 PPR motifs, similar to other proteins of this family that were identified in mutants defected in chloroplast gene expression. To understand the molecular mechanism of how HCF152 modulates chloroplast gene expression, the molecular and biochemical properties should be revealed. To this end, HCF152 and several truncated versions were produced in bacteria and analyzed for RNA-binding and protein-protein interaction. It was found that two HCF152 polypeptides bind to form a homodimer, and that this binding is impaired by a single amino acid substitute near the carboxyl terminus, replacing leucine with proline. Recombinant HCF152 bound with higher affinity RNA molecules, resembling the petB exon-intron junctions, as well as several other molecules. The highest affinity was found to RNA composed of the poly(A) sequence. When truncated proteins composed of different numbers of PPR motifs were analyzed for RNA-binding, it was found that two PPR motifs were required for RNA-binding, but had very low affinity. The affinity to RNA increased significantly when proteins composed of more PPR motifs were analyzed, displaying the highest affinity with the full-length protein composed of 12 PPR motifs. Together, our data characterized the nuclear-encoded HCF152 to be a chloroplast RNA-binding protein that may be involved in the processing or stabilization of the petB transcript by binding to the exon-intron junctions.
Arabidopsis↗
Association of glycolate oxidation with photosynthetic electron transport in plant and algal chloroplasts.
Photosynthetic carbon metabolism is initiated by ribulose-bisphosphate carboxylase/oxygenase (Rubisco), which uses both CO2 and O2 as substrates. One 2-phosphoglycolate (P-glycolate) molecule is produced for each O2 molecule fixed. P-glycolate has been considered to be metabolized exclusively via the oxidative photosynthetic carbon cycle. This paper reports an additional pathway for P-glycolate and glycolate metabolism in the chloroplasts. Light-dependent glycolate or P-glycolate oxidation by osmotically shocked chloroplasts from the algae Dunaliella or spinach leaves was measured by three electron acceptors, methyl viologen (MV), potassium ferricyanide, or dichloroindophenol. Glycolate oxidation was assayed with 3-(3,4)-dichlorophenyl)-1,1-dimethylurea (DCMU) as oxygen uptake in the presence of MV at a rate of 9 mol per mg of chlorophyll per h. Washed thylakoids from spinach leaves oxidized glycolate at a rate of 22 mol per mg of chlorophyll per h. This light-dependent oxidation was inhibited completely by SHAM, an inhibitor of quinone oxidoreductase, and 75% by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), which inhibits electron transfer from plastoquinone to the cytochrome b6f complex. SHAM stimulated severalfold glycolate excretion by algal cells, Dunaliella or Chlamydomonas, and by isolated Dunaliella chloroplasts. Glycolate and P-glycolate were oxidized about equally well to glyoxylate and phosphate. On the basis of results of inhibitor action, the possible site which accepts electrons from glycolate or P-glycolate is a quinone after the DCMU site but before the DBMIB site. This glycolate oxidation is a light-dependent, SHAM-sensitive, glycolate-quinone oxidoreductase system that is associated with photosynthetic electron transport in the chloroplasts.
Journal Article↗
EPR, electron spin echo envelope modulation, and electron nuclear double resonance studies of the 2Fe2S centers of the 2-halobenzoate 1,2-dioxygenase from Burkholderia (Pseudomonas) cepacia 2CBS.
The 2-halobenzoate 1,2-dioxygenase from Burkholderia (Pseudomonas) cepacia 2CBS (Fetzner, S., Müller, R., and Lingens, F. (1992) J. Bacteriol. 174, 279-290) contains both a ferredoxin-type and a Rieske-type 2Fe2S center. These two significantly different 2Fe2S clusters were characterized with respect to their EPR spectra, electrochemical properties (Rieske-type cluster with gz = 2.025, gy = 1.91, gx = 1.79, gav = 1.91, Em = -125 +/- 10 mV; ferredoxin-type center with gz = 2.05, gy = 1.96, gx = 1.89, gav = 1.97, Em = -200 +/- 10 mV) and pH dependence thereof. X band electron spin echo envelope modulation and electron nuclear double resonance spectroscopy was applied to study the interaction of the Rieske-type center of the 2-halobenzoate 1,2-dioxygenase with 14N and 1H nuclei in the vicinity of the 2Fe2S cluster. The results are compared to those obtained on the Rieske protein of the cytochrome b6f complex (Em = +320 mV) and the water-soluble ferredoxin (Em = -430 mV) of spinach chloroplasts, as typical representatives of the gav = 1.91 and gav = 1.96 class of 2Fe2S centers. Properties common to all Rieske-type clusters and those restricted to the respective centers in bacterial oxygenases are discussed.
Burkholderia cepacia↗
Sequencing covalent modifications of membrane proteins.
A number of strategies have successfully extended plant proteomics into the bilayer domain. Important benefits can be afforded by including a well-resolved intact protein mass spectrum alongside peptide identification experiments; recent studies of thylakoid membranes have yielded new information on the primary structure and covalent post-translational modification of many of the integral proteins. Intact mass proteomics is advancing through the development of core technologies in separations and mass spectrometry, with the goal of providing comprehensive primary structure coverage that includes transmembrane domains with reliable measurements of protein abundance and turnover. To address the limitations associated with separation technologies such as 2D-gel electrophoresis, alternative systems are being investigated and 2D-liquid chromatography of thylakoid membrane proteins, using both denaturing and non-denaturing first dimensions, has been successful, extending separation space and providing intact protein solutions for electrospray-ionization mass spectrometry and top-down proteomics. High-resolution conventional, and Fourier-transform, mass spectrometry is bringing increasing resolution to tandem mass spectrometry allowing for 'top-down' mass spectrometry of intact proteins. Thus the core chromatographic technologies already developed for intact mass proteomics of integral membrane proteins also allow their 'top-down' analysis. Thylakoid membrane proteins with one and two transmembrane helices have been analysed demonstrating the ability of collision-activated dissociation (CAD) to sequence through transmembrane domains. When analysed by top-down proteomics, the small subunits of the cytochrome b6f complex from Arabidopsis thylakoids reveal novel post-transcriptional/translational modifications including the presence of glutamic acid at position 2 of PetL instead of the proline residue predicted from the gene sequence.
Chromatography, Agarose↗
Crystal structures of wild-type and mutant plastocyanins from a higher plant, Silene.
Plastocyanin functions as an electron carrier between the cytochrome b6f complex and photosystem I. The crystal structures of the wild-type and E43K/D44K double mutant from the higher plant, Silene, have been determined at 2.0 and 1.75 A resolution, respectively. The wild-type plastocyanin comprises two monomers per asymmetric unit, one of which shows the unusually great distance between the copper ion and the Ndelta1 atom of H87 because of the hydrogen bond network formation between H87 and symmetry-related G10. The root mean square deviation for Ca atoms between the wild-type and mutant plastocyanins is 0.44 A, however, the electrostatic potential maps of their molecular surfaces are remarkably different. The low electron-transfer rate in the E43K/D44K mutant results from the hindrance of electrostatic interactions, not from the structural change due to the mutation.
Copper↗
The Chlamydomonas reinhardtii organellar genomes respond transcriptionally and post-transcriptionally to abiotic stimuli.
The Chlamydomonas reinhardtii plastid and mitochondrial transcriptomes were surveyed for changes in RNA profiles resulting from growth in 12 culture conditions representing 8 abiotic stimuli. Organellar RNA abundance exhibited marked changes during nutrient stress and exposure to UV light, as revealed by both RNA gel blot and DNA microarray analyses. Of particular note were large increases in tufA and clpP transcript abundance during nutrient limitation. Phosphate and sulfur limitation resulted in the most global, yet opposite, effects on organellar RNA abundance, changes that were dissected further using run-on transcription assays. Removal of sulfate from the culture medium, which is known to reduce photosynthesis, resulted in 2-fold to 10-fold decreases in transcription rates, which were reflected in lower RNA abundance. The decrease in transcriptional activity was completely reversible and recovered to twice the control level after sulfate replenishment. Conversely, phosphate limitation resulted in a twofold to threefold increase in RNA abundance that was found to be a post-transcriptional effect, because it could be accounted for by increased RNA stability. This finding is consistent with the known metabolic slowdown under phosphate stress. Additionally, inhibitor studies suggested that unlike those in higher plants, Chlamydomonas chloroplasts lack a nucleus-encoded plastid RNA polymerase. The apparently single type of polymerase could contribute to the rapid and genome-wide transcriptional responses observed within the chloroplast.
Animals↗
Efficient spore synthesis in Bacillus subtilis depends on the CcdA protein.
CcdA is known to be required for the synthesis of c-type cytochromes in Bacillus subtilis, but the exact function of this membrane protein is not known. We show that CcdA also plays a role in spore synthesis. The expression of ccdA and the two downstream genes yneI and yneJ was analyzed. There is a promoter for each gene, but there is only one transcription terminator, located after the yneJ gene. The promoter for ccdA was found to be weak and was active mainly during the transition from exponential growth to stationary phase. The promoters for yneI and yneJ were both active in the exponential growth phase. The levels of the CcdA and YneJ proteins in the membrane were consistent with the observed promoter activities. The ccdA promoter activity was independent of whether the ccdA-yneI-yneJ gene products were absent or overproduced in the cell. It is shown that the four known cytochromes c in B. subtilis and the YneI and YneJ proteins are not required for sporulation. The combined data from analysis of sporulation-specific sigma factor activity, resistance properties of spores, and spore morphology indicate that CcdA deficiency affects stage V in sporulation. We conclude that CcdA, YneI, and YneJ are functionally unrelated proteins and that the role of CcdA in cytochrome c and spore synthesis probably relates to sulfhydryl redox chemistry on the outer surface of the cytoplasmic membrane.
Amino Acid Sequence↗
Introduction to the membrane protein reviews: the interplay of structure, dynamics, and environment in membrane protein function.
In our review, we introduce an organizational scheme for membrane protein function. It is the relationship between structure, dynamics, and environment that endows the membrane and its constituents with remarkable sensitivity and robustness. Our understanding begins with landmark advances like those presented in the following chapters. Membrane proteins are notoriously difficult to study, and so the work presented here on the ADP/ATP carrier [Nury et al. ( 2 )], rhodopsin [Palczewski ( 24 )], and the cytochrome b6f complex [Cramer et al. ( 35 )] represents incredible progress in this now blossoming field.
Humans↗
The cytochrome bc complex (menaquinone:cytochrome c reductase) in Bacillus subtilis has a nontraditional subunit organization.
We have identified an operon in Bacillus subtilis, designated qcr, that is thought to encode a quinone: cytochrome c reductase. Northern (RNA blot) analysis suggests a tricistronic operon. The operon is located at about 200 degrees on the B. subtilis map. Disruption of the operon leads to loss of a 22-kDa cytochrome c from membrane preparations. The structure of the putative protein products of the qcr operon suggests a protein complex that is closely related to but distinct from known cytochrome bc1 and b6f complexes, which catalyze electron transfer from a quinol to a c-type cytochrome or to plastocyanin. QcrA is similar to Rieske-type iron-sulfur proteins; QcrB is similar in size and sequence to b-type cytochromes from b6f complexes; and QcrC has a novel structure that resembles a fusion of a subunit IV (found in b6f complexes) to a cytochrome c. Transcription of the operon is induced at the end of exponential growth from a sigma A-like promoter. This transition state induction appears to be dependent on the downregulation of abrB expression, which is mediated by Spo0A activation. As bacteria move from the transition state into sporulation, transcription of the operon is reduced in a sigma F-dependent manner.
Amino Acid Sequence↗
Site-directed photochemical coupling of cytochrome b6f-associated chlorophyll.
Cytochrome b(6)f complexes contain a molecule of chlorophyll a (Chla), which, in Chlamydomonas reinhardtii, can be exchanged for extraneous chlorophyll during protracted incubation of the purified complex in detergent solution. The specificity of the site and its location in the complex have been studied by photochemical coupling and circular dichroism spectroscopy. Following substitution of the original chlorophyll with [(3)H]Chla, the complex was irradiated in the Soret absorption band of Chla to complete bleaching and the amount of radioactivity covalently bound to each b(6)f subunit determined. Strong labeling was found to be associated with cytochrome f. The labeling originates from [(3)H]Chla molecules bound to a slowly exchanging site and showing the properties of the endogenous Chl, not from molecules dissolved in the detergent belt surrounding the complex. Chlorophyll b (Chlb) can compete with Chla, albeit with a lower affinity. Irradiation of [(3)H]Chlb introduced into the slowly exchanging site yielded the same labeling pattern that was observed with [(3)H]Chla. Proteolytic cleavage showed [(3)H]Chla labeling to be strictly restricted to the C-terminal region of cytochrome f. Circular dichroism spectra of the native complex revealed a bilobed signal characteristic of excitonic interaction between chlorophylls. The structural and evolutionary implications of these findings are discussed.
Animals↗
Chimeric fusions of subunit IV and PetL in the b6f complex of Chlamydomonas reinhardtii: structural implications and consequences on state transitions.
The cytochrome b(6)f complex of Chlamydomonas reinhardtii contains four large subunits and at least three small ones, PetG, PetL, and PetM, whose role and location are unknown. Chimeric proteins have been constructed, in which the C terminus of subunit IV is fused to either one or the other of the two putative N termini of PetL. Biochemical and functional analysis of the chimeras together with mass spectrometry analysis of the wild-type (WT) complex led to the following conclusions: (i) neither a free subunit IV C terminus nor a free PetL N terminus is required for assembly of the b(6)f complex; (ii) the first AUG codon in the sequence of the gene petL is used for initiation; (iii) the N terminus of WT PetL lies in the lumen; (iv) in the WT complex, the N terminus of PetL and the C terminus of subunit IV are within reach of each other; (v) the purified b(6)f complex from C. reinhardtii contains an eighth, hitherto unrecognized subunit, PetN; and (vi) the ability to perform state transitions is lost in the chimeric mutants, although (vii) the Q-cycle is unaffected. A structural hypothesis is presented to account for this peculiar phenotype.
Amino Acid Sequence↗
Proteomic studies of the thylakoid membrane of Synechocystis sp. PCC 6803.
Purified thylakoid membranes from the cyanobacterium Synechocystis sp. PCC 6803 were used for the first time in proteomic studies. The membranes were prepared by a combination of sucrose density centrifugation and aqueous polymer two-phase partitioning. In total, 76 different proteins were identified from 2- and 1-D gels by MALDI-TOF MS analysis. Twelve of the identified proteins have a predicted Sec/Tat signal peptide. Fourteen of the proteins were known, or predicted to be, integral membrane proteins. Among the proteins identified were subunits of the well-characterized thylakoid membrane constituents Photosystem I and II, ATP synthase, cytochrome b6f-complex, NADH dehydrogenase, and phycobilisome complex. In addition, novel thylakoid membrane proteins, both integral and peripheral were found, including enzymes involved in protein folding and pigment biosynthesis. The latter were the chlorophyll biosynthesis enzymes, light-dependent protochlorophyllide reductase and geranylgeranyl reductase as well as phytoene desaturase involved in carotenoid biosynthesis and a water-soluble carotenoid-binding protein. Interestingly, in view of the protein sorting mechanism in cyanobacteria, one of the two signal peptidases type I of Synechocystis was found in the thylakoid membrane, whereas the second one has been identified previously in the plasma membrane. Sixteen proteins are hypothetical proteins with unknown function.
Chloroplast Proton-Translocating ATPases↗
Studies of the cytochrome subunits of menaquinone:cytochrome c reductase (bc complex) of Bacillus subtilis. Evidence for the covalent attachment of heme to the cytochrome b subunit.
The menaquinone:cytochrome c reductase, or bc complex, of Bacillus subtilis belongs to a third class of bc-type complex, distinct from the bc1 and b6f classes. Using a mutagenesis approach, we demonstrate that the cytochrome b (QcrB) and c (QcrC) subunits of the complex give rise to bands at 22 and 29 kDa, respectively, after denaturing electrophoresis; that both subunits are required for proper complex assembly and/or stability; and that both subunits retain one heme molecule under denaturing conditions. This unusual property of a b-type cytochrome was investigated further. We present evidence for the existence of a covalent linkage between the polypeptide and heme bH and of an important role for Cys43 in binding of heme bH. It is proposed that heme is also covalently attached to the cytochrome b subunit of b6f complexes of chloroplasts and cyanobacteria.
Amino Acid Sequence↗
Scanning transmission electron microscopy study of the molecular mass of amphipol/cytochrome b6f complexes.
The composition and mass of complexes between Chlamydomonas reinhardtii cytochrome b6f and low molecular mass amphipathic polymers ('amphipols') have been studied using biochemical analysis and scanning transmission electron microscopy at liquid helium temperature (cryo-STEM). Cytochrome b6f was trapped by amphipols either under its native 14-meric state or as a delipidated, lighter form. A good consistency was observed between the masses of either form calculated from their biochemical composition and those determined by cryo-STEM. These data show that association with amphipols preserved the original original state of the protein in detergent solution. Complexation with amphipols appears to facilitate preparation of the samples and mass determination by cryo-STEM as compared to conventional solubilization with detergents.
Animals↗