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William A. Cramer - One of the best experts on this subject based on the ideXlab platform.
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trans membrane signaling in photosynthetic state transitions redox and structure dependent interaction in vitro between stt7 kinase and the Cytochrome B6f Complex
Journal of Biological Chemistry, 2016Co-Authors: Sandeep Singh, Saif S Hasan, Stanislav D Zakharov, Sejuti Naurin, Whitaker Cohn, Julian P Whitelegge, William A. CramerAbstract:Trans-membrane signaling involving a serine/threonine kinase (Stt7 in Chlamydomonas reinhardtii) directs light energy distribution between the two photosystems of oxygenic photosynthesis. Oxidation of plastoquinol mediated by the Cytochrome B6f Complex on the electrochemically positive side of the thylakoid membrane activates the kinase domain of Stt7 on the trans (negative) side, leading to phosphorylation and redistribution ("state transition") of the light-harvesting chlorophyll proteins between the two photosystems. The molecular description of the Stt7 kinase and its interaction with the Cytochrome B6f Complex are unknown or unclear. In this study, Stt7 kinase has been cloned, expressed, and purified in a heterologous host. Stt7 kinase is shown to be active in vitro in the presence of reductant and purified as a tetramer, as determined by analytical ultracentrifugation, electron microscopy, and electrospray ionization mass spectrometry, with a molecular weight of 332 kDa, consisting of an 83.41-kDa monomer. Far-UV circular dichroism spectra show Stt7 to be mostly α-helical and document a physical interaction with the B6f Complex through increased thermal stability of Stt7 secondary structure. The activity of wild-type Stt7 and its Cys-Ser mutant at positions 68 and 73 in the presence of a reductant suggest that the enzyme does not require a disulfide bridge for its activity as suggested elsewhere. Kinase activation in vivo could result from direct interaction between Stt7 and the B6f Complex or long-range reduction of Stt7 by superoxide, known to be generated in the B6f Complex by quinol oxidation.
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photo induced oxidation of the uniquely liganded heme f in the Cytochrome B6f Complex of oxygenic photosynthesis
Physical Chemistry Chemical Physics, 2016Co-Authors: Adrien Chauvet, Rachna Agarwal, Andre Al Haddad, Frank Van Mourik, William A. CramerAbstract:The ultrafast behavior of the ferrous heme f from the Cytochrome B6f Complex of oxygenic photosynthesis is revealed by means of transient absorption spectroscopy. Benefiting from the use of microfluidic technologies for handling the sample as well as from a complementary frame-by-frame analysis of the heme dynamics, the different relaxation mechanisms from vibrationally excited states are disentangled and monitored via the shifts of the heme α-absorption band. Under 520 nm laser excitation, about 85% of the heme f undergoes pulse-limited photo-oxidation (<100 fs), with the electron acceptor being most probably one of the adjacent aromatic amino acid residues. After charge recombination in 5.3 ps, the residual excess energy is dissipated in 3.6 ps. In a parallel pathway, the remaining 15% of the hemes directly relax from their excited state in 2.5 ps. In contrast to a vast variety of heme-proteins, including the homologous heme c1 from the Cytochrome bc1 Complex, there is no evidence that heme f photo-dissociates from its axial ligands. Due to its unique binding, with histidine and an unusual tyrosine as axial ligands, the heme f exemplifies a dependence of ultrafast dynamics on the structural environment.
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the enigmatic chloroplast stt7 kinase trans membrane function with Cytochrome B6f Complex in situ kinase activity in vitro
Biophysical Journal, 2016Co-Authors: Sandeep Singh, Whitaker Cohn, Julian P Whitelegge, S S Hasan, William A. CramerAbstract:The distribution of light energy between the two photosystems in oxygenic photosynthesis is regulated in the alga, C. reinhardtii, by a 754 residue State Transition Kinase, Stt7, which is unique in using a trans-membrane topology to carry out its signaling function (1). It is activated by oxidation of plastoquinol on the electrochemically positive, lumen side of the Cytochrome B6f Complex (2), and phosphorylates the major light-harvesting chlorophyll protein II (3) on the opposite side of the membrane (1). Stt7, which binds to cyt B6f (1), requires a disulfide bond on the p-side proximal to the quinol oxidation site for its activation (1, 3). In the present study, Stt7 has been cloned in E. coli and purified as a soluble protein whose monomer mass, determined by mass spectrometry is 79,501. On Clear Native PAGE, Stt7 runs close to a position expected for a heptamer. Purified Stt7 has significant redox-dependent kinase activity in vitro. The demonstration of in vitro activity, the absence of a documented membrane bound state, and the small stoichiometry (circa 1:20) of interaction with the B6f Complex (1), suggest that reaction of the kinase with the LHCII occurs through a membrane-peripheral domain of the B6f Complex. (1) Lemeille, S. et al. (2009) PLoS Biology 7, e1000045; (2) Vener, A. V. et al. (1997) PNAS, 94, 1585-1590. (3) Millner, P. et al., J. Biol. Chem., 257, 1736-1742, 1982. Support from NIHGMS-038323.
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structure function of the Cytochrome b 6 f lipoprotein Complex
2016Co-Authors: William A. Cramer, Saif S HasanAbstract:The Cytochrome B6f Complex (f, derived from “leaf” in Latin, “folium,” or French, “feuille”, see D. S. Bendall, this volume) is responsible for the proton-coupled electron transfer reactions that link the two light-trapping photosystem reaction centers in the electron transport chain of oxygenic photosynthesis, and contains the rate-limiting step of the entire electron transport chain. The understanding of the functions of the intra-membrane Cytochrome B6f Complex in its role as the central electron transport Complex in the photosynthetic electron transport chain has greatly increased in recent years because of the solving of the atomic structure of the Complex, as well as its extrinsic electron transfer domains solved separately, by X-ray crystallographic analysis. The electron transfer and proton translocation functions of the Cytochrome B6f Complex are discussed in a context that focuses on function in the context of structure. The structure considerations are based on the recently determined high resolution (2.50 A) crystal structure (PDB, protein data base, accession 4OGQ). The Complex consists of a 280 kDa hetero-oligomeric intra-membrane lipo-protein Complex containing, per monomer, 13 trans-membrane α-helices and seven prosthetic groups, the latter consisting of five hemes, one chlorophyll a molecule and one β-carotene. In addition, each monomer contains 23 lipid binding sites, the function of which crosses into a completely new area of structure-function, both for the B6f Complex and for the entire broader field of membrane proteins. Considering energy-transducing membrane protein Complexes, the B6f Complex, along with other hetero-oligomeric electron transfer membrane proteins provides a unique perspective in the broad field of membrane protein structure-function that encompasses transport and channel proteins, and extends beyond photosynthesis and bioenergetics to biomedicine. The additional perspective arises from the fact that the function of the Cytochrome Complexes, along with those of other redox proteins, can be assayed quantitatively through various high resolution spectroscopies.
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internal lipid architecture of the hetero oligomeric Cytochrome B6f Complex
Structure, 2014Co-Authors: Saif S Hasan, William A. CramerAbstract:Summary The role of lipids in the assembly, structure, and function of hetero-oligomeric membrane protein Complexes is poorly understood. The dimeric photosynthetic Cytochrome b 6 f Complex, a 16-mer of eight distinct subunits and 26 transmembrane helices, catalyzes transmembrane proton-coupled electron transfer for energy storage. Using a 2.5 A crystal structure of the dimeric Complex, we identified 23 distinct lipid-binding sites per monomer. Annular lipids are proposed to provide a connection for super-Complex formation with the photosystem-I reaction center and the LHCII kinase enzyme for transmembrane signaling. Internal lipids mediate crosslinking to stabilize the domain-swapped iron-sulfur protein subunit, dielectric heterogeneity within intermonomer and intramonomer electron transfer pathways, and dimer stabilization through lipid-mediated intermonomer interactions. This study provides a complete structure analysis of lipid-mediated functions in a multi-subunit membrane protein Complex and reveals lipid sites at positions essential for assembly and function.
Saif S Hasan - One of the best experts on this subject based on the ideXlab platform.
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trans membrane signaling in photosynthetic state transitions redox and structure dependent interaction in vitro between stt7 kinase and the Cytochrome B6f Complex
Journal of Biological Chemistry, 2016Co-Authors: Sandeep Singh, Saif S Hasan, Stanislav D Zakharov, Sejuti Naurin, Whitaker Cohn, Julian P Whitelegge, William A. CramerAbstract:Trans-membrane signaling involving a serine/threonine kinase (Stt7 in Chlamydomonas reinhardtii) directs light energy distribution between the two photosystems of oxygenic photosynthesis. Oxidation of plastoquinol mediated by the Cytochrome B6f Complex on the electrochemically positive side of the thylakoid membrane activates the kinase domain of Stt7 on the trans (negative) side, leading to phosphorylation and redistribution ("state transition") of the light-harvesting chlorophyll proteins between the two photosystems. The molecular description of the Stt7 kinase and its interaction with the Cytochrome B6f Complex are unknown or unclear. In this study, Stt7 kinase has been cloned, expressed, and purified in a heterologous host. Stt7 kinase is shown to be active in vitro in the presence of reductant and purified as a tetramer, as determined by analytical ultracentrifugation, electron microscopy, and electrospray ionization mass spectrometry, with a molecular weight of 332 kDa, consisting of an 83.41-kDa monomer. Far-UV circular dichroism spectra show Stt7 to be mostly α-helical and document a physical interaction with the B6f Complex through increased thermal stability of Stt7 secondary structure. The activity of wild-type Stt7 and its Cys-Ser mutant at positions 68 and 73 in the presence of a reductant suggest that the enzyme does not require a disulfide bridge for its activity as suggested elsewhere. Kinase activation in vivo could result from direct interaction between Stt7 and the B6f Complex or long-range reduction of Stt7 by superoxide, known to be generated in the B6f Complex by quinol oxidation.
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structure function of the Cytochrome b 6 f lipoprotein Complex
2016Co-Authors: William A. Cramer, Saif S HasanAbstract:The Cytochrome B6f Complex (f, derived from “leaf” in Latin, “folium,” or French, “feuille”, see D. S. Bendall, this volume) is responsible for the proton-coupled electron transfer reactions that link the two light-trapping photosystem reaction centers in the electron transport chain of oxygenic photosynthesis, and contains the rate-limiting step of the entire electron transport chain. The understanding of the functions of the intra-membrane Cytochrome B6f Complex in its role as the central electron transport Complex in the photosynthetic electron transport chain has greatly increased in recent years because of the solving of the atomic structure of the Complex, as well as its extrinsic electron transfer domains solved separately, by X-ray crystallographic analysis. The electron transfer and proton translocation functions of the Cytochrome B6f Complex are discussed in a context that focuses on function in the context of structure. The structure considerations are based on the recently determined high resolution (2.50 A) crystal structure (PDB, protein data base, accession 4OGQ). The Complex consists of a 280 kDa hetero-oligomeric intra-membrane lipo-protein Complex containing, per monomer, 13 trans-membrane α-helices and seven prosthetic groups, the latter consisting of five hemes, one chlorophyll a molecule and one β-carotene. In addition, each monomer contains 23 lipid binding sites, the function of which crosses into a completely new area of structure-function, both for the B6f Complex and for the entire broader field of membrane proteins. Considering energy-transducing membrane protein Complexes, the B6f Complex, along with other hetero-oligomeric electron transfer membrane proteins provides a unique perspective in the broad field of membrane protein structure-function that encompasses transport and channel proteins, and extends beyond photosynthesis and bioenergetics to biomedicine. The additional perspective arises from the fact that the function of the Cytochrome Complexes, along with those of other redox proteins, can be assayed quantitatively through various high resolution spectroscopies.
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internal lipid architecture of the hetero oligomeric Cytochrome B6f Complex
Structure, 2014Co-Authors: Saif S Hasan, William A. CramerAbstract:Summary The role of lipids in the assembly, structure, and function of hetero-oligomeric membrane protein Complexes is poorly understood. The dimeric photosynthetic Cytochrome b 6 f Complex, a 16-mer of eight distinct subunits and 26 transmembrane helices, catalyzes transmembrane proton-coupled electron transfer for energy storage. Using a 2.5 A crystal structure of the dimeric Complex, we identified 23 distinct lipid-binding sites per monomer. Annular lipids are proposed to provide a connection for super-Complex formation with the photosystem-I reaction center and the LHCII kinase enzyme for transmembrane signaling. Internal lipids mediate crosslinking to stabilize the domain-swapped iron-sulfur protein subunit, dielectric heterogeneity within intermonomer and intramonomer electron transfer pathways, and dimer stabilization through lipid-mediated intermonomer interactions. This study provides a complete structure analysis of lipid-mediated functions in a multi-subunit membrane protein Complex and reveals lipid sites at positions essential for assembly and function.
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dielectric heterogeneity in the Cytochrome B6f Complex
Biophysical Journal, 2014Co-Authors: Stanislav D Zakharov, Saif S Hasan, Sergei Savikhin, Adrien Chauvet, Valentyn Stadnytsky, William A. CramerAbstract:Electron transfer in the dimeric Cytochrome B6f Complex, which includes four b-type hemes organized as two pairs in symmetric monomers, was studied by simultaneous measurement of the kinetics of heme reduction by dithionite and an associated amplitude increase of Soret band split circular dichroism (CD) spectra diagnostic of heme-heme exciton interactions, for which similar kinetics were determined. Based on inter-heme distances and orientations from crystal structures of the Complex, the increase in the split CD signal is dominated by interaction between the two intra-monomer b-hemes, located on the electrochemically negative and positive sides of the Complex, whose midpoint oxidation-reduction potentials, Em, determined by titrations of isolated Complex, differ by 75-100 mV. Kinetics are fit best by preferential reduction of the intra-monomer heme pair. Equilibration of transferred electrons would, however, predict preferential reduction of the two higher potential hemes, one in each monomer. Heterogeneity of the dielectric constant is implied, a consequence of structure inhomogeneity, and/or dielectric reorganization in response to electron transfer. The largest dielectric constant exists between the intra-monomer b-hemes, resulting in a lower energy state of the reduced intra-monomer heme pair relative to any other heme pair.View Large Image | View Hi-Res Image | Download PowerPoint Slide
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mechanism of enhanced superoxide production in the Cytochrome b 6 f Complex of oxygenic photosynthesis
Biochemistry, 2013Co-Authors: Danas Baniulis, Saif S Hasan, Jason T Stofleth, William A. CramerAbstract:The specific rate of superoxide (O2 − ) production in the purified active crystallizable Cytochrome B6f Complex, normalized to the rate of electron transport, has been found to be more than an order of magnitude greater than that measured in isolated yeast respiratory bc1 Complex. The biochemical and structural basis for the enhanced production of O2 − in the Cytochrome B6f Complex compared to that in the bc1 Complex is discussed. The higher rate of superoxide production in the B6f Complex could be a consequence of an increased residence time of plastosemiquinone/ plastoquinol in its binding niche near the Rieske protein iron−sulfur cluster, resulting from (i) occlusion of the quinone portal by the phytyl chain of the unique bound chlorophyll, (ii) an altered environment of the proton-accepting glutamate believed to be a proton acceptor from semiquinone, or (iii) a more negative redox potential of the heme bp on the electrochemically positive side of the Complex. The enhanced rate of superoxide production in the B6f Complex is physiologically significant as the chloroplast-generated reactive oxygen species (ROS) functions in the regulation of excess excitation energy, is a source of oxidative damage inflicted during photosynthetic reactions, and is a major source of ROS in plant cells. Altered levels of ROS production are believed to convey redox signaling from the organelle to the cytosol and nucleus.
Huamin Zhang - One of the best experts on this subject based on the ideXlab platform.
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purification and crystallization of the cyanobacterial Cytochrome B6f Complex
Methods of Molecular Biology, 2011Co-Authors: Danas Baniulis, Huamin Zhang, Taisiya Zakharova, Saif S Hasan, William A. CramerAbstract:The Cytochrome B6f Complex from the filamentous cyanobacteria (Mastigocladus laminosus, Nostoc sp. PCC 7120) and spinach chloroplasts has been purified as a homo-dimer. Electrospray ionization mass spectroscopy showed the monomer to contain eight and nine subunits, respectively, and dimeric masses of 217.1, 214.2, and 286.5 kDa for M. laminosus, Nostoc, and the Complex from spinach. The core subunits containing or interacting with redox-active prosthetic groups are petA (Cytochrome f), B (Cytochrome b6, C (Rieske iron-sulfur protein), D (subunit IV), with protein molecular weights of 31.8-32.3, 24.7-24.9, 18.9-19.3, and 17.3-17.5 kDa, and four small 3.2-4.2 kDa polypeptides petG, L, M, and N. A ninth polypeptide, the 35 kDa petH (FNR) polypeptide in the spinach Complex, was identified as ferredoxin:NADP reductase (FNR), which binds to the Complex tightly at a stoichiometry of approx 0.8/cytf. The spinach Complex contains diaphorase activity diagnostic of FNR and is active in facilitating ferredoxin-dependent electron transfer from NADPH to the Cytochrome B6f Complex. The purified Cytochrome B6f Complex contains stoichiometrically bound chlorophyll a and β-carotene at a ratio of approximately one molecule of each per Cytochrome f. It also contains bound lipid and detergent, indicating seven lipid-binding sites per monomer. Highly purified Complexes are active for approximately 1 week after isolation, transferring 200-300 electrons/cytf s. The M. laminosus Complex was shown to be subject to proteolysis and associated loss of activity if incubated for more than 1 week at room temperature. The Nostoc Complex is more resistant to proteolysis. Addition of pure synthetic lipid to the cyanobacterial Complex, which is mostly delipidated by the isolation procedure, allows rapid formation of large (≥0.2 mm) crystals suitable for X-ray diffraction analysis and structure determination. The crystals made from the cyanobacterial Complex diffract to 3.0 A with R values of 0.222 and 0.230 for M. laminosus and Nostoc, respectively. It has not yet been possible to obtain crystals of the B6f Complex from any plant source, specifically spinach or pea, perhaps because of incomplete binding of FNR or other peripheral polypeptides. Well diffracting crystals have been obtained from the green alga, Chlamydomonas reinhardtii (ref. 10).
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purification and crystallization of the cyanobacterial Cytochrome b 6 f Complex
Methods of Molecular Biology, 2011Co-Authors: Danas Baniulis, Huamin Zhang, Taisiya Zakharova, Saif S Hasan, William A. CramerAbstract:The Cytochrome B6f Complex from the filamentous cyanobacteria (Mastigocladus laminosus, Nostoc sp. PCC 7120) and spinach chloroplasts has been purified as a homo-dimer. Electrospray ionization mass spectroscopy showed the monomer to contain eight and nine subunits, respectively, and dimeric masses of 217.1, 214.2, and 286.5 kDa for M. laminosus, Nostoc, and the Complex from spinach. The core subunits containing or interacting with redox-active prosthetic groups are petA (Cytochrome f), B (Cytochrome b6, C (Rieske iron-sulfur protein), D (subunit IV), with protein molecular weights of 31.8-32.3, 24.7-24.9, 18.9-19.3, and 17.3-17.5 kDa, and four small 3.2-4.2 kDa polypeptides petG, L, M, and N. A ninth polypeptide, the 35 kDa petH (FNR) polypeptide in the spinach Complex, was identified as ferredoxin:NADP reductase (FNR), which binds to the Complex tightly at a stoichiometry of approx 0.8/cytf. The spinach Complex contains diaphorase activity diagnostic of FNR and is active in facilitating ferredoxin-dependent electron transfer from NADPH to the Cytochrome B6f Complex. The purified Cytochrome B6f Complex contains stoichiometrically bound chlorophyll a and β-carotene at a ratio of approximately one molecule of each per Cytochrome f. It also contains bound lipid and detergent, indicating seven lipid-binding sites per monomer. Highly purified Complexes are active for approximately 1 week after isolation, transferring 200-300 electrons/cytf s. The M. laminosus Complex was shown to be subject to proteolysis and associated loss of activity if incubated for more than 1 week at room temperature. The Nostoc Complex is more resistant to proteolysis. Addition of pure synthetic lipid to the cyanobacterial Complex, which is mostly delipidated by the isolation procedure, allows rapid formation of large (≥0.2 mm) crystals suitable for X-ray diffraction analysis and structure determination. The crystals made from the cyanobacterial Complex diffract to 3.0 A with R values of 0.222 and 0.230 for M. laminosus and Nostoc, respectively. It has not yet been possible to obtain crystals of the B6f Complex from any plant source, specifically spinach or pea, perhaps because of incomplete binding of FNR or other peripheral polypeptides. Well diffracting crystals have been obtained from the green alga, Chlamydomonas reinhardtii (ref. 10).
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structure function of the Cytochrome B6f Complex
Photochemistry and Photobiology, 2008Co-Authors: Danas Baniulis, Huamin Zhang, Saif S Hasan, Eiki Yamashita, William A. CramerAbstract:The structure and function of the Cytochrome b6 f Complex is considered in the context of recent crystal structures of the Complex as an eight subunit, 220 kDa symmetric dimeric Complex obtained from the thermophilic cyanobacterium, Mastigocladus laminosus, and the green alga, Chlamydomonas reinhardtii. A major problem confronted in crystallization of the cyanobacterial Complex, proteolysis of three of the subunits, is discussed along with initial efforts to identify the protease. The evolution of these Cytochrome Complexes is illustrated by conservation of the hydrophobic heme-binding transmembrane domain of the cyt b polypeptide between b6 f and bc1 Complexes, and the rubredoxin-like membrane proximal domain of the Rieske [2Fe-2S] protein. Pathways of coupled electron and proton transfer are discussed in the framework of a modified Q cycle, in which the heme cn, not found in the bc1 Complex, but electronically tightly coupled to the heme bn of the b6 f Complex, is included. Crystal structures of the cyanobacterial Complex with the quinone analogue inhibitors, NQNO or tridecylstigmatellin, show the latter to be ligands of heme cn, implicating heme cn as an n-side plastoquinone reductase. Existing questions include (a) the details of the shuttle of: (i) the [2Fe-2S] protein between the membrane-bound PQH2 electron ⁄ H + donor and the Cytochrome f acceptor to complete the p-side electron transfer circuit; (ii) PQ ⁄ PQH2 between n- and p-sides of the Complex across the intermonomer quinone exchange cavity, through the narrow portal connecting the cavity with the p-side [2Fe-2S] niche; (b) the role of the n-side of the b6 f Complex and heme cn in regulation of the relative rates of noncyclic and cyclic electron transfer. The likely presence of cyclic electron transport in the b6 f Complex, and of heme cn in the firmicute bc Complex suggests the concept that hemes bn-cn define a branch point in bc Complexes that can support electron transport pathways that differ in detail from the Q cycle supported by the bc1 Complex.
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structure of the Cytochrome B6f Complex n side donor pathway to the plastoquinone pool
2008Co-Authors: Danas Baniulis, Huamin Zhang, Eiki Yamashita, William A. CramerAbstract:Three Prosthetic Groups, Heme CN, Chl A, And β-Carotene Were Identified In Crystal Structures Of The Cytochrome B 6 F Complex In The Thermophilic Cyanobacterium, M. Laminosus (Kurisu Et Al. 2003) And The Green Alga, C. Reinhardtii (Stroebel Et Al. 2003). The Functions Of These Groups Are Still Not Understood. A Native Structure Of The Cytochrome B 6 F Complex From The Thermophilic Cyanobacterium, M. Laminosus, Was Obtained From Crystals Grown With Divalent Cadmium (Pdb Accession: 2E74; (Yamashita Et Al. 2007) ). One Cd2+ Binding Site Bridges His143 Of Cytochrome F And The Acidic Residue, Glu75, Of Cyt B 6; (Ii) A Second Site Has Three Identified Ligands, Asp58 (Subunit Iv), Glu3 (Petg Subunit) And Glu4 (Petm Subunit). Binding Sites Of Quinone Analogue Inhibitors Map The Transfer Pathway Of The Lipophilic Quinone Across The Complex. Two Sites Were Found For The Chromone Ring Of The Tridecyl-Stigmatellin (Tds) Quinone Analogue Inhibitor, One Near The P-Side [2Fe-2S] Cluster (Pdb: 2E76). A Second Tds Site Faces The Quinone Exchange Cavity As An Axial Ligand Of Heme C N. A Similar Binding Site As An Axial Ligand To Heme C N Was Found For The N-Side Quinone Analogue Inhibitor, Nqno (Pdb: 2E75). Binding Of These Inhibitors Required Their Addition Before That Of The Lipid Used To Facilitate Crystallization. Binding Of Nqno And Tds As Axial Ligands To Heme C N Implies That C N Utilizes Plastoquinone As A Natural Ligand, Thus Defining An N-Side Electron Transfer Compleand Pq In Thex Consisting Of Hemes B N, C N, And Pq In The Reduction Pathway Of Pq In The Cavity. Strong Coupling Of Hemes Bn And Cn Suggests A Mechanism For 2 Electron Reduction Of Pq, Thus Avoiding The Generation Of Plastosemiquionone And Reactive Oxygen Species. The Nqno Binding Site Explains Several Experimental Observations Associated With Its Inhibitory Action: A Negative Shift In The Heme C N EM (Alric Et Al. 2005), Increased Amplitude Of Light-Induced Reduction Of Heme B N (Jones And Whitmarsh 1988; Furbacher Et Al. 1989), And G Value Shifts In The Epr Spectrum Attributed To Interaction Between Hemes C N And B N (Zatsman Et Al. 2006; Baymann Et Al. 2007). These Structures Suggest Pathways For H+ Uptake And Potential Site(S) Of Ferredoxin Binding.
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heme heme interactions in the Cytochrome B6f Complex epr spectroscopy and correlation with structure
Journal of the American Chemical Society, 2006Co-Authors: Anna I Zatsman, William A. Cramer, Huamin Zhang, William A Gunderson, Michael P HendrichAbstract:Cytochrome B6f of oxygenic photosynthesis was studied using multifrequency, multimode EPR Spectroscopy. Frequency dependent signals above g = 4.3, and the observation of parallel-mode signals, are indicative of spin interactions in the Complex. We demonstrate the presence of an exchange interaction between the unique high-spin heme cn and a nearby low-spin heme bn, and show that a quinone analog NQNO binds at or near to heme cn. The two hemes remain spin coupled upon the binding of NQNO, though strength of interaction decreases significantly. The electronic coupling implies that the heme bn/cn pair could function as a unit to facilitate 2-electron reduction of plastoquionone without generation of an energetically unfavorable semiquinone intermediate.
Danas Baniulis - One of the best experts on this subject based on the ideXlab platform.
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mechanism of enhanced superoxide production in the Cytochrome b 6 f Complex of oxygenic photosynthesis
Biochemistry, 2013Co-Authors: Danas Baniulis, Saif S Hasan, Jason T Stofleth, William A. CramerAbstract:The specific rate of superoxide (O2 − ) production in the purified active crystallizable Cytochrome B6f Complex, normalized to the rate of electron transport, has been found to be more than an order of magnitude greater than that measured in isolated yeast respiratory bc1 Complex. The biochemical and structural basis for the enhanced production of O2 − in the Cytochrome B6f Complex compared to that in the bc1 Complex is discussed. The higher rate of superoxide production in the B6f Complex could be a consequence of an increased residence time of plastosemiquinone/ plastoquinol in its binding niche near the Rieske protein iron−sulfur cluster, resulting from (i) occlusion of the quinone portal by the phytyl chain of the unique bound chlorophyll, (ii) an altered environment of the proton-accepting glutamate believed to be a proton acceptor from semiquinone, or (iii) a more negative redox potential of the heme bp on the electrochemically positive side of the Complex. The enhanced rate of superoxide production in the B6f Complex is physiologically significant as the chloroplast-generated reactive oxygen species (ROS) functions in the regulation of excess excitation energy, is a source of oxidative damage inflicted during photosynthetic reactions, and is a major source of ROS in plant cells. Altered levels of ROS production are believed to convey redox signaling from the organelle to the cytosol and nucleus.
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quinone dependent proton transfer pathways in the photosynthetic Cytochrome B6f Complex
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Saif S Hasan, Danas Baniulis, Eiki Yamashita, William A. CramerAbstract:As much as two-thirds of the proton gradient used for transmembrane free energy storage in oxygenic photosynthesis is generated by the Cytochrome B6f Complex. The proton uptake pathway from the electrochemically negative (n) aqueous phase to the n-side quinone binding site of the Complex, and a probable route for proton exit to the positive phase resulting from quinol oxidation, are defined in a 2.70-A crystal structure and in structures with quinone analog inhibitors at 3.07 A (tridecyl-stigmatellin) and 3.25-A (2-nonyl-4-hydroxyquinoline N-oxide) resolution. The simplest n-side proton pathway extends from the aqueous phase via Asp20 and Arg207 (Cytochrome b6 subunit) to quinone bound axially to heme cn. On the positive side, the heme-proximal Glu78 (subunit IV), which accepts protons from plastosemiquinone, defines a route for H+ transfer to the aqueous phase. These pathways provide a structure-based description of the quinone-mediated proton transfer responsible for generation of the transmembrane electrochemical potential gradient in oxygenic photosynthesis.
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increased superoxide production in the Cytochrome B6f Complex a function for the enigmatic chlorophyll a
Biophysical Journal, 2013Co-Authors: Danas Baniulis, Saif S Hasan, Jason T Stofleth, William A. CramerAbstract:The structural basis for superoxide production in Cytochrome bc Complexes is relevant to understanding the mechanism of generation of deleterious reactive oxygen species and partition of electron transfer in the branched quinol oxidation pathway in Cytochrome bc Complexes. The specific rate of superoxide production, normalized to the the electron transfer rate, was determined for the yeast Cytochrome bc1 Complex (provided by B. L. Trumpower) and the Cytochrome B6f Complex from spinach thylakoid membranes and cyanobacteria. Although electron transfer rates were comparable in bc1 and B6f Complexes, the specific rate of superoxide production was 10-20 fold greater in the B6f Complex. Whereas antimycin A, a specific n-side quinone analogue inhibitor of the Cytochrome bc1 Complex, caused a large increase in the superoxide production rate of the bc1 Complex, no comparable effect was found for NQNO, an n-side quinone analogue inhibitor in B6f, as defined by spectrophotometry and a crystal structure. These differences between bc1 and B6f Complexes imply an increase in branching ratio for reduction by plasto-semiquinone of O2 to O2-, relative to reduction of heme bp for trans-membrane electron transfer. The change in branching ratio is ascribed to a longer semiquinone residence time in the p-side binding niche, due to steric restriction of the quinone binding site by the chlorophyll phytyl chain, as seen in a crystal structure. The presence of this phytyl chain can be seen to result in a smaller accessible volume for binding sites of a p-side quinone analogue inhibitor. The longer residence time of quinol/semiquinone would facilitate trans-membrane signaling, e.g., activation of n-side LHC kinase (NIH GM-38323).
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an anhydrous proton transfer pathway in the Cytochrome B6f Complex
Biophysical Journal, 2013Co-Authors: Saif S Hasan, Danas Baniulis, Eiki Yamashita, William A. CramerAbstract:Cytochrome B6f, a hetero-oligomeric energy transducing membrane protein Complex, catalyzes proton-coupled electron transfer reactions of the substrate quinone to generate as much as two-thirds of the total proton gradient used for ATP synthesis in oxygenic photosynthesis. Proton transfer pathways within B6f have remained unidentified due to limitations of crystallographic resolution1,2,3,4,5. using new crystallographic information based on a 2.70 A crystal structure of the native Complex, and structures obtained in the presence of quinone analogue inhibitors, tridecyl-stigmatellin (TDS) and 2-nonyl-4-hydroxyquinoline-N-oxide (NQNO) (resolution 3.07 A and 3.25 A respectively) seen in close proximity to heme cn on the electrochemically negative (n) side of the B6f Complex, an anhydrous proton uptake pathway is defined that delivers protons from the n-side aqueous phase to plastoquinone bound at heme cn. A hydrated channel is identified on the electrochemically positive (p) side of the Complex, that may provide a route for proton exit to the p-side aqueous phase. These n- and p-side pathways contribute to the first complete description of quinone-mediated trans-membrane proton transfer.1. Kurisu et al. 2003, 2Stroebel et al. 2003, 3Yan et al. 2006, 4Yamashita et al. 2007, 5Baniulis et al. 2009View Large Image | View Hi-Res Image | Download PowerPoint Slide
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purification and crystallization of the cyanobacterial Cytochrome B6f Complex
Methods of Molecular Biology, 2011Co-Authors: Danas Baniulis, Huamin Zhang, Taisiya Zakharova, Saif S Hasan, William A. CramerAbstract:The Cytochrome B6f Complex from the filamentous cyanobacteria (Mastigocladus laminosus, Nostoc sp. PCC 7120) and spinach chloroplasts has been purified as a homo-dimer. Electrospray ionization mass spectroscopy showed the monomer to contain eight and nine subunits, respectively, and dimeric masses of 217.1, 214.2, and 286.5 kDa for M. laminosus, Nostoc, and the Complex from spinach. The core subunits containing or interacting with redox-active prosthetic groups are petA (Cytochrome f), B (Cytochrome b6, C (Rieske iron-sulfur protein), D (subunit IV), with protein molecular weights of 31.8-32.3, 24.7-24.9, 18.9-19.3, and 17.3-17.5 kDa, and four small 3.2-4.2 kDa polypeptides petG, L, M, and N. A ninth polypeptide, the 35 kDa petH (FNR) polypeptide in the spinach Complex, was identified as ferredoxin:NADP reductase (FNR), which binds to the Complex tightly at a stoichiometry of approx 0.8/cytf. The spinach Complex contains diaphorase activity diagnostic of FNR and is active in facilitating ferredoxin-dependent electron transfer from NADPH to the Cytochrome B6f Complex. The purified Cytochrome B6f Complex contains stoichiometrically bound chlorophyll a and β-carotene at a ratio of approximately one molecule of each per Cytochrome f. It also contains bound lipid and detergent, indicating seven lipid-binding sites per monomer. Highly purified Complexes are active for approximately 1 week after isolation, transferring 200-300 electrons/cytf s. The M. laminosus Complex was shown to be subject to proteolysis and associated loss of activity if incubated for more than 1 week at room temperature. The Nostoc Complex is more resistant to proteolysis. Addition of pure synthetic lipid to the cyanobacterial Complex, which is mostly delipidated by the isolation procedure, allows rapid formation of large (≥0.2 mm) crystals suitable for X-ray diffraction analysis and structure determination. The crystals made from the cyanobacterial Complex diffract to 3.0 A with R values of 0.222 and 0.230 for M. laminosus and Nostoc, respectively. It has not yet been possible to obtain crystals of the B6f Complex from any plant source, specifically spinach or pea, perhaps because of incomplete binding of FNR or other peripheral polypeptides. Well diffracting crystals have been obtained from the green alga, Chlamydomonas reinhardtii (ref. 10).
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functional characterization of the small regulatory subunit petp from the Cytochrome B6f Complex in thermosynechococcus elongatus
The Plant Cell, 2014Co-Authors: Sascha Rexroth, Marc M Nowaczyk, Dorothea Rexroth, Sebastian Veit, Nicole Plohnke, Kai U Cormann, Matthias RögnerAbstract:The cyanobacterial Cytochrome B6f Complex is central for the coordination of photosynthetic and respiratory electron transport and also for the balance between linear and cyclic electron transport. The development of a purification strategy for a highly active dimeric B6f Complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 enabled characterization of the structural and functional role of the small subunit PetP in this Complex. Moreover, the efficient transformability of this strain allowed the generation of a ΔpetP mutant. Analysis on the whole-cell level by growth curves, photosystem II light saturation curves, and P700+ reduction kinetics indicate a strong decrease in the linear electron transport in the mutant strain versus the wild type, while the cyclic electron transport via photosystem I and Cytochrome B6f is largely unaffected. This reduction in linear electron transport is accompanied by a strongly decreased stability and activity of the isolated ΔpetP Complex in comparison with the dimeric wild-type Complex, which binds two PetP subunits. The distinct behavior of linear and cyclic electron transport may suggest the presence of two distinguishable pools of Cytochrome B6f Complexes with different functions that might be correlated with superComplex formation.
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petg and petn but not petl are essential subunits of the Cytochrome B6f Complex from synechocystis pcc 6803
Research in Microbiology, 2007Co-Authors: Dirk Schneider, Thomas Volkmer, Matthias RögnerAbstract:Abstract The Cytochrome B6f Complex consists of four large core subunits and an additional four low molecular weight subunits, the function of which is elusive thus far. Here we sought to determine whether small subunits PetG, PetL, and PetN are essential for a cyanobacterial Cytochrome B6f Complex. We found that only PetL is dispensable, whereas PetG and PetN appear to be essential. Possible roles of the small Cytochrome B6f Complex subunits are discussed, and observations from our study are compared with previous findings.
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Heterogeneous Rieske proteins in the Cytochrome B6f Complex of Synechocystis PCC6803
The Journal of biological chemistry, 2002Co-Authors: Dirk Schneider, Andreas Seidler, Sven Skrzypczak, Stefan Anemüller, Christian Schmidt, Matthias RögnerAbstract:Abstract The completely sequenced genome of the cyanobacterium Synechocystis PCC6803 contains three open reading frames, petC1, petC2, and petC3,encoding putative Rieske iron-sulfur proteins. After heterologous overexpression, all three gene products have been characterized and shown to be Rieske proteins as typified by sequence analysis and EPR spectroscopy. Two of the overproduced proteins contained already incorporated iron-sulfur clusters, whereas the third one formed unstable aggregates, in which the FeS cluster had to be reconstituted after refolding of the denatured protein. Although EPR spectroscopy showed typical FeS signals for all Rieske proteins, an unusual low midpoint potential was revealed for PetC3 by EPR redox titration. Detailed characterization of Synechocystismembranes indicated that all three Rieske proteins are expressed under physiological conditions. Both for PetC1 and PetC3 the association with the thylakoid membrane was shown, and both could be identified, although in different amounts, in the isolated Cytochromeb 6 f Complex. The considerably lower redox potential determined for PetC3 indicates heterogeneous Cytochromeb 6 f Complexes inSynechocystis and suggests still to be established alternative electron transport routes.
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a regulatory role of the petm subunit in a cyanobacterial Cytochrome B6f Complex
Journal of Biological Chemistry, 2001Co-Authors: Dirk Schneider, Stephan Berry, Peter R Rich, Andreas Seidler, Matthias RögnerAbstract:To investigate the function of the PetM subunit of the Cytochrome B6f Complex, the petM gene encoding this subunit was inactivated by insertional mutagenesis in the cyanobacterium Synechocystis PCC 6803. Complete segregation of the mutant reveals a nonessential function of PetM for the structure and function of the Cytochrome B6f Complex in this organism. Photosystem I, photosystem II, and the Cytochrome B6f Complex still function normally in the petM- mutant as judged by Cytochrome f re-reduction and oxygen evolution rates. In contrast to the wild type, however, the content of phycobilisomes and photosystem I as determined from 77 K fluorescence spectra is reduced in the petM- strain. Furthermore, whereas under anaerobic conditions the kinetics of Cytochrome f re-reduction are identical, under aerobic conditions these kinetics are slower in the petM- strain. Fluorescence induction measurements indicate that this is due to an increased plastoquinol oxidase activity in the mutant, causing the plastoquinone pool to be in a more oxidized state under aerobic dark conditions. The finding that the activity of the Cytochrome B6f Complex itself is unchanged, whereas the stoichiometry of other protein Complexes has altered, suggests an involvement of the PetM subunit in regulatory processes mediated by the Cytochrome B6f Complex.
