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William A Cramer - One of the best experts on this subject based on the ideXlab platform.
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structural and functional contributions of lipids to the stability and activity of the photosynthetic Cytochrome B6f lipoprotein complex
Journal of Biological Chemistry, 2019Co-Authors: Satarupa Bhaduri, Huamin Zhang, Satchal K Erramilli, William A CramerAbstract:The photosynthetic Cytochrome B6f complex, a homodimer containing eight distinct subunits and 26 transmembrane helices per monomer, catalyzes proton-coupled electron transfer across the thylakoid membrane. The 2.5-A-resolution structure of the complex from the cyanobacterium Nostoc sp. revealed the presence of 23 lipid-binding sites per monomer. Although the crystal structure of the Cytochrome B6f from a plant source has not yet been solved, the identities of the lipids present in a plant B6f complex have previously been determined, indicating that the predominant lipid species are monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), phosphatidylglycerol (PG), and sulfoquinovosyldiacylglycerol (SQDG). Despite the extensive structural analyses of B6f-lipid interactions, the basis of the stabilization by lipids remains poorly understood. In the present study, we report on the effect of individual lipids on the structural and functional integrity of the B6f complex, purified from Spinacea oleracea It was found that (i) galactolipids (MGDG, DGDG, and SQDG) and phospholipids dilinolenoyl-phosphatidylglycerol (DLPG), 1,2-dioleoylphosphatidylglycerol (DOPG), and 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC) structurally stabilize the complex to varying degrees; (ii) SQDG has a major role in stabilizing the dimeric complex; (iii) the B6f complex is stabilized by incorporation into nanodiscs or bicelles; (iv) removal of bound phospholipid by phospholipase A2 inactivates the Cytochrome complex; and (v) activity can be restored significantly by the addition of the anionic lipid PG, which is attributed to stabilization of the quinone portal and the hinge region of the iron-sulfur protein.
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structure function of the Cytochrome B6f lipoprotein complex a scientific odyssey and personal perspective
Photosynthesis Research, 2019Co-Authors: William A CramerAbstract:Structure–function studies of the Cytochrome B6f complex, the central hetero-oligomeric membrane protein complex in the electron transport chain of oxygenic photosynthesis, which formed the basis for a high-resolution (2.5 A) crystallographic solution of the complex, are described. Structure–function differences between the structure of subunits of the bc complexes, B6f, and bc1 from mitochondria and photosynthetic bacteria, which are often assumed to function identically, are discussed. Major differences which suggest that quinone-dependent electron transport pathways can vary in B6f and bc1 complexes are as follows: (a) an additional c-type heme, cn, and bound single copies of chlorophyll a and β-carotene in the B6f complex; and (b) a cyclic electron transport pathway that encompasses the B6f and PSI reaction center complexes. The importance of including lipid in crystallization of the Cytochrome complex, or with any hetero-oligomeric membrane protein complex, is emphasized, and consequences to structure–function of B6f being a lipoprotein complex discussed, including intra-protein dielectric heterogeneity and resultant pathways of trans-membrane electron transport. The role of the B6f complex in trans-membrane signal transduction from reductant generated on the p-side of the electron transport chain to the regulation of light energy to the two photosystems by trans-side phosphorylation of the light-harvesting chlorophyll protein is presented. Regarding structure aspects relevant to plastoquinol-quinone entrance-egress: (i) modification of the p-side channel for plastoquinone access to the iron-sulfur protein would change the rate-limiting step in electron transport; (ii) the narrow niche for entry of plastoquinol into B6f from the PSII reaction center complex would seem to require close proximity between the complexes.
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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.
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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role of domain swapping in the hetero oligomeric Cytochrome B6f lipoprotein complex
Biochemistry, 2015Co-Authors: Rachna Agarwal, Saif S Hasan, Julian P Whitelegge, Jason T Stofleth, Christopher M Ryan, Ladonna M Jones, David M Kehoe, William A CramerAbstract:Domain swapping that contributes to the stability of biologically crucial multisubunit complexes has been implicated in protein oligomerization. In the case of membrane protein assemblies, domain swapping of the iron–sulfur protein (ISP) subunit occurs in the hetero-oligomeric Cytochrome B6f and bc1 complexes, which are organized as symmetric dimers that generate the transmembrane proton electrochemical gradient utilized for ATP synthesis. In these complexes, the ISP C-terminal predominantly β-sheet extrinsic domain containing the redox-active [2Fe-2S] cluster resides on the electrochemically positive side of each monomer in the dimeric complex. This domain is bound to the membrane sector of the complex through an N-terminal transmembrane α-helix that is “swapped’ to the other monomer of the complex where it spans the complex and the membrane. Detailed analysis of the function and structure of the B6f complex isolated from the cyanobacterium Fremyella diplosiphon SF33 shows that the domain-swapped ISP str...
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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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lipid induced conformational changes within the Cytochrome B6f complex of oxygenic photosynthesis
Biochemistry, 2013Co-Authors: Saif S Hasan, Eiki Yamashita, Jason T Stofleth, William A CramerAbstract:Cytochrome B6f catalyzes quinone redox reactions within photosynthetic membranes to generate a transmembrane proton electrochemical gradient for ATP synthesis. A key step involves the transfer of an electron from the [2Fe-2S] cluster of the iron–sulfur protein (ISP) extrinsic domain to the Cytochrome f heme across a distance of 26 A, which is too large for competent electron transfer but could be bridged by translation–rotation of the ISP. Here we report the first crystallographic evidence of significant motion of the ISP extrinsic domain. It is inferred that extensive crystallographic disorder of the ISP extrinsic domain indicates conformational flexibility. The ISP disorder observed in this structure, in contrast to the largely ordered ISP structure observed in the B6f complex supplemented with neutral lipids, is attributed to electrostatic interactions arising from anionic lipids.
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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.
Matthew P Johnson - One of the best experts on this subject based on the ideXlab platform.
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Cytochrome B6f - Orchestrator of photosynthetic electron transfer.
Biochimica et biophysica acta. Bioenergetics, 2021Co-Authors: Lorna A. Malone, Matthew S. Proctor, Andrew Hitchcock, C. Neil Hunter, Matthew P JohnsonAbstract:Abstract Cytochrome B6f (cytB6f) lies at the heart of the light-dependent reactions of oxygenic photosynthesis, where it serves as a link between photosystem II (PSII) and photosystem I (PSI) through the oxidation and reduction of the electron carriers plastoquinol (PQH2) and plastocyanin (Pc). A mechanism of electron bifurcation, known as the Q-cycle, couples electron transfer to the generation of a transmembrane proton gradient for ATP synthesis. CytB6f catalyses the rate-limiting step in linear electron transfer (LET), is pivotal for cyclic electron transfer (CET) and plays a key role as a redox-sensing hub involved in the regulation of light-harvesting, electron transfer and photosynthetic gene expression. Together, these characteristics make cytB6f a judicious target for genetic manipulation to enhance photosynthetic yield, a strategy which already shows promise. In this review we will outline the structure and function of cytB6f with a particular focus on new insights provided by the recent high-resolution map of the complex from Spinach.
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cryo em structure of the spinach Cytochrome B6f complex at 3 6 angstrom resolution
Nature, 2019Co-Authors: Lorna A. Malone, Andrew Hitchcock, Pu Qian, Guy E Mayneord, David A Farmer, Rebecca F Thompson, David J K Swainsbury, Neil A Ranson, C N Hunter, Matthew P JohnsonAbstract:The Cytochrome b6 f (cytb6 f ) complex has a central role in oxygenic photosynthesis, linking electron transfer between photosystems I and II and converting solar energy into a transmembrane proton gradient for ATP synthesis1–3. Electron transfer within cytb6 f occurs via the quinol (Q) cycle, which catalyses the oxidation of plastoquinol (PQH2) and the reduction of both plastocyanin (PC) and plastoquinone (PQ) at two separate sites via electron bifurcation2. In higher plants, cytb6 f also acts as a redox-sensing hub, pivotal to the regulation of light harvesting and cyclic electron transfer that protect against metabolic and environmental stresses3. Here we present a 3.6 A resolution cryo-electron microscopy (cryo-EM) structure of the dimeric cytb6 f complex from spinach, which reveals the structural basis for operation of the Q cycle and its redox-sensing function. The complex contains up to three natively bound PQ molecules. The first, PQ1, is located in one cytb6 f monomer near the PQ oxidation site (Qp) adjacent to haem bp and chlorophyll a. Two conformations of the chlorophyll a phytyl tail were resolved, one that prevents access to the Qp site and another that permits it, supporting a gating function for the chlorophyll a involved in redox sensing. PQ2 straddles the intermonomer cavity, partially obstructing the PQ reduction site (Qn) on the PQ1 side and committing the electron transfer network to turnover at the occupied Qn site in the neighbouring monomer. A conformational switch involving the haem cn propionate promotes two-electron, two-proton reduction at the Qn site and avoids formation of the reactive intermediate semiquinone. The location of a tentatively assigned third PQ molecule is consistent with a transition between the Qp and Qn sites in opposite monomers during the Q cycle. The spinach cytb6 f structure therefore provides new insights into how the complex fulfils its catalytic and regulatory roles in photosynthesis. A 3.6 A resolution cryo-electron microscopy structure of the dimeric Cytochrome B6f complex from spinach reveals the structural basis for operation of the quinol cycle and its redox-sensing function.
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single molecule study of redox control involved in establishing the spinach plastocyanin Cytochrome B6f electron transfer complex
Biochimica et Biophysica Acta, 2019Co-Authors: Guy E Mayneord, Cvetelin Vasilev, Neil C Hunter, Lorna A. Malone, David J K Swainsbury, Matthew P JohnsonAbstract:Small diffusible redox proteins play a ubiquitous role in bioenergetic systems, facilitating electron transfer (ET) between membrane bound complexes. Sustaining high ET turnover rates requires that the association between extrinsic and membrane-bound partners is highly specific, yet also sufficiently weak to promote rapid post-ET separation. In oxygenic photosynthesis the small soluble electron carrier protein plastocyanin (Pc) shuttles electrons between the membrane integral Cytochrome B6f (cytB6f) and photosystem I (PSI) complexes. Here we use peak-force quantitative nanomechanical mapping (PF-QNM) atomic force microscopy (AFM) to quantify the dynamic forces involved in transient interactions between cognate ET partners. An AFM probe functionalised with Pc molecules is brought into contact with cytB6f complexes, immobilised on a planar silicon surface. PF-QNM interrogates the unbinding force of the cytB6f-Pc interactions at the single molecule level with picoNewton force resolution and on a time scale comparable to the ET time in vivo (ca. 120 μs). Using this approach, we show that although the unbinding force remains unchanged the interaction frequency increases over five-fold when Pc and cytB6f are in opposite redox states, so complementary charges on the cytB6f and Pc cofactors likely contribute to the electrostatic forces that initiate formation of the ET complex. These results suggest that formation of the docking interface is under redox state control, which lowers the probability of unproductive encounters between Pc and cytB6f molecules in the same redox state, ensuring the efficiency and directionality of this central reaction in the ‘Z-scheme’ of photosynthetic ET.
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single molecule study of redox control involved in establishing the spinach plastocyanin Cytochrome B6f electron transfer complex
Biochimica et Biophysica Acta, 2019Co-Authors: Guy E Mayneord, Cvetelin Vasilev, Neil C Hunter, Lorna A. Malone, David J K Swainsbury, Matthew P JohnsonAbstract:Small diffusible redox proteins play a ubiquitous role in bioenergetic systems, facilitating electron transfer (ET) between membrane bound complexes. Sustaining high ET turnover rates requires that the association between extrinsic and membrane-bound partners is highly specific, yet also sufficiently weak to promote rapid post-ET separation. In oxygenic photosynthesis the small soluble electron carrier protein plastocyanin (Pc) shuttles electrons between the membrane integral Cytochrome B6f (cytB6f) and photosystem I (PSI) complexes. Here we use peak-force quantitative nanomechanical mapping (PF-QNM) atomic force microscopy (AFM) to quantify the dynamic forces involved in transient interactions between cognate ET partners. An AFM probe functionalised with Pc molecules is brought into contact with cytB6f complexes, immobilised on a planar silicon surface. PF-QNM interrogates the unbinding force of the cytB6f-Pc interactions at the single molecule level with picoNewton force resolution and on a time scale comparable to the ET time in vivo (ca. 120 μs). Using this approach, we show that although the unbinding force remains unchanged the interaction frequency increases over five-fold when Pc and cytB6f are in opposite redox states, so complementary charges on the cytB6f and Pc cofactors likely contribute to the electrostatic forces that initiate formation of the ET complex. These results suggest that formation of the docking interface is under redox state control, which lowers the probability of unproductive encounters between Pc and cytB6f molecules in the same redox state, ensuring the efficiency and directionality of this central reaction in the ‘Z-scheme’ of photosynthetic ET.
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nanodomains of Cytochrome B6f and photosystem ii complexes in spinach grana thylakoid membranes
The Plant Cell, 2014Co-Authors: Matthew P Johnson, Cvetelin Vasilev, John D Olsen, Neil C HunterAbstract:The Cytochrome B6f (cytB6f) complex plays a central role in photosynthesis, coupling electron transport between photosystem II (PSII) and photosystem I to the generation of a transmembrane proton gradient used for the biosynthesis of ATP. Photosynthesis relies on rapid shuttling of electrons by plastoquinone (PQ) molecules between PSII and cytB6f complexes in the lipid phase of the thylakoid membrane. Thus, the relative membrane location of these complexes is crucial, yet remains unknown. Here, we exploit the selective binding of the electron transfer protein plastocyanin (Pc) to the lumenal membrane surface of the cytB6f complex using a Pc-functionalized atomic force microscope (AFM) probe to identify the position of cytB6f complexes in grana thylakoid membranes from spinach (Spinacia oleracea). This affinity-mapping AFM method directly correlates membrane surface topography with Pc-cytB6f interactions, allowing us to construct a map of the grana thylakoid membrane that reveals nanodomains of colocalized PSII and cytB6f complexes. We suggest that the close proximity between PSII and cytB6f complexes integrates solar energy conversion and electron transfer by fostering short-range diffusion of PQ in the protein-crowded thylakoid membrane, thereby optimizing photosynthetic efficiency.
Huamin Zhang - One of the best experts on this subject based on the ideXlab platform.
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structural and functional contributions of lipids to the stability and activity of the photosynthetic Cytochrome B6f lipoprotein complex
Journal of Biological Chemistry, 2019Co-Authors: Satarupa Bhaduri, Huamin Zhang, Satchal K Erramilli, William A CramerAbstract:The photosynthetic Cytochrome B6f complex, a homodimer containing eight distinct subunits and 26 transmembrane helices per monomer, catalyzes proton-coupled electron transfer across the thylakoid membrane. The 2.5-A-resolution structure of the complex from the cyanobacterium Nostoc sp. revealed the presence of 23 lipid-binding sites per monomer. Although the crystal structure of the Cytochrome B6f from a plant source has not yet been solved, the identities of the lipids present in a plant B6f complex have previously been determined, indicating that the predominant lipid species are monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), phosphatidylglycerol (PG), and sulfoquinovosyldiacylglycerol (SQDG). Despite the extensive structural analyses of B6f-lipid interactions, the basis of the stabilization by lipids remains poorly understood. In the present study, we report on the effect of individual lipids on the structural and functional integrity of the B6f complex, purified from Spinacea oleracea It was found that (i) galactolipids (MGDG, DGDG, and SQDG) and phospholipids dilinolenoyl-phosphatidylglycerol (DLPG), 1,2-dioleoylphosphatidylglycerol (DOPG), and 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC) structurally stabilize the complex to varying degrees; (ii) SQDG has a major role in stabilizing the dimeric complex; (iii) the B6f complex is stabilized by incorporation into nanodiscs or bicelles; (iv) removal of bound phospholipid by phospholipase A2 inactivates the Cytochrome complex; and (v) activity can be restored significantly by the addition of the anionic lipid PG, which is attributed to stabilization of the quinone portal and the hinge region of the iron-sulfur protein.
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JL: Evolution of photosynthesis: time-independent structure of the Cytochrome B6f complex
2014Co-Authors: William A Cramer, Huamin Zhang, Genji Kurisu, Jiusheng Yan, Janet L. SmithAbstract:ABSTRACT: Structures of the Cytochrome B6f complex obtained from the thermophilic cyanobacterium Mastigocladus laminosus and the green alga Chlamydomonas reinhardtii, whose appearance in evolution is separated by 109 years, are almost identical. Two monomers with a molecular weight of 110 000, containing eight subunits and seven natural prosthetic groups, are separated by a large lipid-containing “quinone exchange cavity”. A unique heme, heme x, that is five-coordinated and high-spin, with no strong field ligand, occupies a position close to intramembrane heme bn. This position is filled by the n-side bound quinone, Qn, in the Cytochrome bc1 complex of the mitochondrial respiratory chain. The structure and position of heme x suggest that it could function in ferredoxin-dependent cyclic electron transport as well as being an intermediate in a quinone cycle mechanism for electron and proton transfer. The significant differences between the cyanobacterial and algal structures are as follows. (i) On the n-side, a plastoquinone molecule is present in the quinone exchange cavity in the cyanobacterial complex, and a sulfolipid is bound in the algal complex at a position corresponding to a synthetic DOPC lipid molecule in the cyanobacterial complex. (ii) On the p-side, in both complexes a quinone analogue inhibitor, TDS, passes through a portal that separates the large cavity from a niche containing the Fe2S2 cluster. However, in the cyanobacterial complex, TDS is in an orientation that is the opposite of its position in the algal structur
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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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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, Huamin Zhang, William A Cramer, 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.
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the single chlorophyll a molecule in the Cytochrome B6f complex unusual optical properties protect the complex against singlet oxygen
Biophysical Journal, 2005Co-Authors: Naranbaatar Dashdorj, Huamin Zhang, William A Cramer, Jiusheng Yan, Hanyoup Kim, Sergei SavikhinAbstract:The Cytochrome B6f complex of oxygenic photosynthesis mediates electron transfer between the reaction centers of photosystems I and II and facilitates coupled proton translocation across the membrane. High-resolution x-ray crystallographic structures (Kurisu et al., 2003; Stroebel et al., 2003) of the Cytochrome B6f complex unambiguously show that a Chl a molecule is an intrinsic component of the Cytochrome B6f complex. Although the functional role of this Chl a is presently unclear (Kuhlbrandt, 2003), an excited Chl a molecule is known to produce toxic singlet oxygen as the result of energy transfer from the excited triplet state of the Chl a to oxygen molecules. To prevent singlet oxygen formation in light-harvesting complexes, a carotenoid is typically positioned within ∼4 A of the Chl a molecule, effectively quenching the triplet excited state of the Chl a. However, in the Cytochrome B6f complex, the β-carotene is too far (≥14 A) from the Chl a for effective quenching of the Chl a triplet excited state. In this study, we propose that in this complex, the protection is at least partly realized through special arrangement of the local protein structure, which shortens the singlet excited state lifetime of the Chl a by a factor of 20–25 and thus significantly reduces the formation of the Chl a triplet state. Based on optical ultrafast absorption difference experiments and structure-based calculations, it is proposed that the Chl a singlet excited state lifetime is shortened due to electron exchange transfer with the nearby tyrosine residue. To our knowledge, this kind of protection mechanism against singlet oxygen has not yet been reported for any other chlorophyll-containing protein complex. It is also reported that the Chl a molecule in the Cytochrome B6f complex does not change orientation in its excited state.
Neil C Hunter - One of the best experts on this subject based on the ideXlab platform.
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single molecule study of redox control involved in establishing the spinach plastocyanin Cytochrome B6f electron transfer complex
Biochimica et Biophysica Acta, 2019Co-Authors: Guy E Mayneord, Cvetelin Vasilev, Neil C Hunter, Lorna A. Malone, David J K Swainsbury, Matthew P JohnsonAbstract:Small diffusible redox proteins play a ubiquitous role in bioenergetic systems, facilitating electron transfer (ET) between membrane bound complexes. Sustaining high ET turnover rates requires that the association between extrinsic and membrane-bound partners is highly specific, yet also sufficiently weak to promote rapid post-ET separation. In oxygenic photosynthesis the small soluble electron carrier protein plastocyanin (Pc) shuttles electrons between the membrane integral Cytochrome B6f (cytB6f) and photosystem I (PSI) complexes. Here we use peak-force quantitative nanomechanical mapping (PF-QNM) atomic force microscopy (AFM) to quantify the dynamic forces involved in transient interactions between cognate ET partners. An AFM probe functionalised with Pc molecules is brought into contact with cytB6f complexes, immobilised on a planar silicon surface. PF-QNM interrogates the unbinding force of the cytB6f-Pc interactions at the single molecule level with picoNewton force resolution and on a time scale comparable to the ET time in vivo (ca. 120 μs). Using this approach, we show that although the unbinding force remains unchanged the interaction frequency increases over five-fold when Pc and cytB6f are in opposite redox states, so complementary charges on the cytB6f and Pc cofactors likely contribute to the electrostatic forces that initiate formation of the ET complex. These results suggest that formation of the docking interface is under redox state control, which lowers the probability of unproductive encounters between Pc and cytB6f molecules in the same redox state, ensuring the efficiency and directionality of this central reaction in the ‘Z-scheme’ of photosynthetic ET.
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single molecule study of redox control involved in establishing the spinach plastocyanin Cytochrome B6f electron transfer complex
Biochimica et Biophysica Acta, 2019Co-Authors: Guy E Mayneord, Cvetelin Vasilev, Neil C Hunter, Lorna A. Malone, David J K Swainsbury, Matthew P JohnsonAbstract:Small diffusible redox proteins play a ubiquitous role in bioenergetic systems, facilitating electron transfer (ET) between membrane bound complexes. Sustaining high ET turnover rates requires that the association between extrinsic and membrane-bound partners is highly specific, yet also sufficiently weak to promote rapid post-ET separation. In oxygenic photosynthesis the small soluble electron carrier protein plastocyanin (Pc) shuttles electrons between the membrane integral Cytochrome B6f (cytB6f) and photosystem I (PSI) complexes. Here we use peak-force quantitative nanomechanical mapping (PF-QNM) atomic force microscopy (AFM) to quantify the dynamic forces involved in transient interactions between cognate ET partners. An AFM probe functionalised with Pc molecules is brought into contact with cytB6f complexes, immobilised on a planar silicon surface. PF-QNM interrogates the unbinding force of the cytB6f-Pc interactions at the single molecule level with picoNewton force resolution and on a time scale comparable to the ET time in vivo (ca. 120 μs). Using this approach, we show that although the unbinding force remains unchanged the interaction frequency increases over five-fold when Pc and cytB6f are in opposite redox states, so complementary charges on the cytB6f and Pc cofactors likely contribute to the electrostatic forces that initiate formation of the ET complex. These results suggest that formation of the docking interface is under redox state control, which lowers the probability of unproductive encounters between Pc and cytB6f molecules in the same redox state, ensuring the efficiency and directionality of this central reaction in the ‘Z-scheme’ of photosynthetic ET.
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probing the local lipid environment of the rhodobacter sphaeroides Cytochrome bc1 and synechocystis sp pcc 6803 Cytochrome B6f complexes with styrene maleic acid
Biochimica et Biophysica Acta, 2017Co-Authors: David J K Swainsbury, Matthew S. Proctor, Andrew Hitchcock, Pu Qian, Michael L Cartron, Elizabeth C Martin, Philip J Jackson, Jeppe Madsen, Steven P Armes, Neil C HunterAbstract:Abstract Intracytoplasmic vesicles (chromatophores) in the photosynthetic bacterium Rhodobacter sphaeroides represent a minimal structural and functional unit for absorbing photons and utilising their energy for the generation of ATP. The Cytochrome bc1 complex (cytbc1) is one of the four major components of the chromatophore alongside the reaction centre-light harvesting 1-PufX core complex (RC-LH1-PufX), the light-harvesting 2 complex (LH2), and ATP synthase. Although the membrane organisation of these complexes is known, their local lipid environments have not been investigated. Here we utilise poly(styrene-alt-maleic acid) (SMA) co-polymers as a tool to simultaneously determine the local lipid environments of the RC-LH1-PufX, LH2 and cytbc1 complexes. SMA has previously been reported to effectively solubilise complexes in lipid-rich membrane regions whilst leaving lipid-poor ordered protein arrays intact. Here we show that SMA solubilises cytbc1 complexes with an efficiency of nearly 70%, whereas solubilisation of RC-LH1-PufX and LH2 was only 10% and 22% respectively. This high susceptibility of cytbc1 to SMA solubilisation is consistent with this complex residing in a locally lipid-rich region. SMA solubilised cytbc1 complexes retain their native dimeric structure and co-purify with 56 ± 6 phospholipids from the chromatophore membrane. We extended this approach to the model cyanobacterium Synechocystis sp. PCC 6803, and show that the Cytochrome B6f complex (cytB6f) and Photosystem II (PSII) complexes are susceptible to SMA solubilisation, suggesting they also reside in lipid-rich environments. Thus, lipid-rich membrane regions could be a general requirement for cytbc1/cytB6f complexes, providing a favourable local solvent to promote rapid quinol/quinone binding and release at the Q0 and Qi sites.
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nanodomains of Cytochrome B6f and photosystem ii complexes in spinach grana thylakoid membranes
The Plant Cell, 2014Co-Authors: Matthew P Johnson, Cvetelin Vasilev, John D Olsen, Neil C HunterAbstract:The Cytochrome B6f (cytB6f) complex plays a central role in photosynthesis, coupling electron transport between photosystem II (PSII) and photosystem I to the generation of a transmembrane proton gradient used for the biosynthesis of ATP. Photosynthesis relies on rapid shuttling of electrons by plastoquinone (PQ) molecules between PSII and cytB6f complexes in the lipid phase of the thylakoid membrane. Thus, the relative membrane location of these complexes is crucial, yet remains unknown. Here, we exploit the selective binding of the electron transfer protein plastocyanin (Pc) to the lumenal membrane surface of the cytB6f complex using a Pc-functionalized atomic force microscope (AFM) probe to identify the position of cytB6f complexes in grana thylakoid membranes from spinach (Spinacia oleracea). This affinity-mapping AFM method directly correlates membrane surface topography with Pc-cytB6f interactions, allowing us to construct a map of the grana thylakoid membrane that reveals nanodomains of colocalized PSII and cytB6f complexes. We suggest that the close proximity between PSII and cytB6f complexes integrates solar energy conversion and electron transfer by fostering short-range diffusion of PQ in the protein-crowded thylakoid membrane, thereby optimizing photosynthetic efficiency.
