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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 b 6 f 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 b 6 f 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 b 6 f 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 b 6 f complex or long-range reduction of Stt7 by superoxide, known to be generated in the b 6 f complex by quinol oxidation., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The cytochrome bc complexes b6f and bc1 catalyze proton-coupled quinol/quinone redox reactions to generate a transmembrane proton electrochemical gradient. Quinol oxidation on the electrochemically positive (p) interface of the complex occurs at the end of a narrow quinol/quinone entry/exit Qp portal, 11 Å long in bc complexes. Superoxide, which has multiple signaling functions, is a by-product of the p-side quinol oxidation. Although the transmembrane core and the chemistry of quinone redox reactions are conserved in bc complexes, the rate of superoxide generation is an order of magnitude greater in the b6f complex, implying that functionally significant differences in structure exist between the b6f and bc1 complexes on the p-side. A unique structure feature of the b6f p-side quinol oxidation site is the presence of a single chlorophyll-a molecule whose function is unrelated to light harvesting. This study describes a cocrystal structure of the cytochrome b6f complex with the quinol analog stigmatellin, which partitions in the Qp portal of the bc1 complex, but not effectively in b6f. It is inferred that the Qp portal is partially occluded in the b6f complex relative to bc1. Based on a discrete molecular-dynamics analysis, occlusion of the Qp portal is attributed to the presence of the chlorophyll phytyl tail, which increases the quinone residence time within the Qp portal and is inferred to be a cause of enhanced superoxide production. This study attributes a novel (to our knowledge), structure-linked function to the otherwise enigmatic chlorophyll-a in the b6f complex, which may also be relevant to intracellular redox signaling., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The role of lipids in the assembly, structure, and function of hetero-oligomeric membrane protein complexes is poorly understood. The dimeric photosynthetic cytochrome b6f 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 Å 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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The cytochrome b6f complex, a member of the cytochrome bc family that mediates energy transduction in photosynthetic and respiratory membranes, is a hetero-oligomeric complex that utilizes two pairs of b-hemes in a symmetric dimer to accomplish trans-membrane electron transfer, quinone oxidation-reduction, and generation of a proton electrochemical potential. Analysis of electron storage in this pathway, utilizing simultaneous measurement of heme reduction, and of circular dichroism (CD) spectra, to assay heme-heme interactions, implies a heterogeneous distribution of the dielectric constants that mediate electrostatic interactions between the four hemes in the complex. Crystallographic information was used to determine the identity of the interacting hemes. The Soret band CD signal is dominated by excitonic interaction between the intramonomer b-hemes, bn and bp, on the electrochemically negative and positive sides of the complex. Kinetic data imply that the most probable pathway for transfer of the two electrons needed for quinone oxidation-reduction utilizes this intramonomer heme pair, contradicting the expectation based on heme redox potentials and thermodynamics, that the two higher potential hemes bn on different monomers would be preferentially reduced. Energetically preferred intramonomer electron storage of electrons on the intramonomer b-hemes is found to require heterogeneity of interheme dielectric constants. Relative to the medium separating the two higher potential hemes bn, a relatively large dielectric constant must exist between the intramonomer b-hemes, allowing a smaller electrostatic repulsion between the reduced hemes. Heterogeneity of dielectric constants is an additional structure-function parameter of membrane protein complexes.

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.

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 Å, 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.

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-Å crystal structure and in structures with quinone analog inhibitors at 3.07 Å (tridecyl-stigmatellin) and 3.25-Å (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 c(n). 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.

Lipid-binding sites and properties were compared in the hetero-oligomeric cytochrome (cyt) b(6)f and the yeast bc(1) complexes that function, respectively, in photosynthetic and respiratory electron transport. Seven lipid-binding sites in the monomeric unit of the dimeric cyanobacterial b(6)f complex overlap four sites in the Chlamydomonas reinhardtii algal b(6)f complex and four in the yeast bc(1) complex. The proposed lipid functions include: (i) interfacial-interhelix mediation between (a) the two 8-subunit monomers of the dimeric complex, (b) between the core domain (cyt b, subunit IV) and the six trans membrane helices of the peripheral domain (cyt f, iron-sulphur protein (ISP), and four small subunits in the boundary 'picket fence'); (ii) stabilization of the ISP domain-swapped trans-membrane helix; (iii) neutralization of basic residues in the single helix of cyt f and of the ISP; (iv) a 'latch' to photosystem I provided by the β-carotene chain protruding through the 'picket fence'; (v) presence of a lipid and chlorophyll a chlorin ring in b(6)f in place of the eighth helix in the bc(1) cyt b polypeptide. The question is posed of the function of the lipid substitution in relation to the evolutionary change between the eight and seven helix structures of the cyt b polypeptide. On the basis of the known n-side activation of light harvesting complex II (LHCII) kinase by the p-side level of plastoquinol, one possibility is that the change was directed by the selective advantage of p- to n-side trans membrane signalling functions in b(6)f, with the lipid either mediating this function or substituting for the trans membrane helix of a signalling protein lost in crystallization.

Considering information in the crystal structures of the cytochrome b(6)f complex relevant to the rate-limiting step in oxygenic photosynthesis, it is enigmatic that electron transport in the complex is not limited by the large distance, approximately 26 Å, between the iron-sulfur cluster (ISP) and its electron acceptor, cytochrome f. This enigma has been explained for the respiratory bc(1) complex by a crystal structure with a greatly shortened cluster-heme c(1) distance, leading to a concept of ISP dynamics in which the ISP soluble domain undergoes a translation-rotation conformation change and oscillates between positions relatively close to the cyt c(1) heme and a membrane-proximal position close to the ubiquinol electron-proton donor. Comparison of cytochrome b(6)f structures shows a variation in cytochrome f heme position that suggests the possibility of flexibility and motion of the extended cytochrome f structure that could entail a transient decrease in cluster-heme f distance. The dependence of cyt f turnover on lumen viscosity is consistent with a role of ISP - cyt f dynamics in determination of the rate-limiting step under conditions of low light intensity. Under conditions of low light intensity and proton electrochemical gradient present, for example, under a leaf canopy, it is proposed that a rate limitation of electron transport in the b(6)f complex may also arise from steric constraints in the entry/exit portal for passage of the plastoquinol and -quinone to/from its oxidation site proximal to the iron-sulfur cluster.

Lipid binding sites and properties are compared in two sub-families of hetero-oligomeric membrane protein complexes known to have similar functions in order to gain further understanding of the role of lipid in the function, dynamics, and assembly of these complexes. Using the crystal structure information for both complexes, we compared the lipid binding properties of the cytochrome b(6)f and bc(1) complexes that function in photosynthetic and respiratory membrane energy transduction. Comparison of lipid and detergent binding sites in the b(6)f complex with those in bc(1) shows significant conservation of lipid positions. Seven lipid binding sites in the cyanobacterial b(6)f complex overlap three natural sites in the Chlamydomonas reinhardtii algal complex and four sites in the yeast mitochondrial bc(1) complex. The specific identity of lipids is different in b(6)f and bc(1) complexes: b(6)f contains sulfoquinovosyldiacylglycerol, phosphatidylglycerol, phosphatidylcholine, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol, whereas cardiolipin, phosphatidylethanolamine, and phosphatidic acid are present in the yeast bc(1) complex. The lipidic chlorophyll a and β-carotene (β-car) in cyanobacterial b(6)f, as well as eicosane in C. reinhardtii, are unique to the b(6)f complex. Inferences of lipid binding sites and functions were supported by sequence, interatomic distance, and B-factor information on interacting lipid groups and coordinating amino acid residues. The lipid functions inferred in the b(6)f complex are as follows: (i) substitution of a transmembrane helix by a lipid and chlorin ring, (ii) lipid and β-car connection of peripheral and core domains, (iii) stabilization of the iron-sulfur protein transmembrane helix, (iv) n-side charge and polarity compensation, and (v) β-car-mediated super-complex with the photosystem I complex., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Aspects of the crystal structures of the hetero-oligomeric cytochrome bc(1) and b(6)f ("bc") complexes relevant to their electron/proton transfer function and the associated redox reactions of the lipophilic quinones are discussed. Differences between the b(6)f and bc(1) complexes are emphasized. The cytochrome bc(1) and b(6)f dimeric complexes diverge in structure from a core of subunits that coordinate redox groups consisting of two bis-histidine coordinated hemes, a heme b(n) and b(p) on the electrochemically negative (n) and positive (p) sides of the complex, the high potential [2Fe-2S] cluster and c-type heme at the p-side aqueous interface and aqueous phase, respectively, and quinone/quinol binding sites on the n- and p-sides of the complex. The bc(1) and b(6)f complexes diverge in subunit composition and structure away from this core. b(6)f Also contains additional prosthetic groups including a c-type heme c(n) on the n-side, and a chlorophyll a and β-carotene. Common structure aspects; functions of the symmetric dimer. (I) Quinone exchange with the bilayer. An inter-monomer protein-free cavity of approximately 30Å along the membrane normal×25Å (central inter-monomer distance)×15Å (depth in the center), is common to both bc(1) and b(6)f complexes, providing a niche in which the lipophilic quinone/quinol (Q/QH(2)) can be exchanged with the membrane bilayer. (II) Electron transfer. The dimeric structure and the proximity of the two hemes b(p) on the electrochemically positive side of the complex in the two monomer units allow the possibility of two alternate routes of electron transfer across the complex from heme b(p) to b(n): intra-monomer and inter-monomer involving electron cross-over between the two hemes b(p). A structure-based summary of inter-heme distances in seven bc complexes, representing mitochondrial, chromatophore, cyanobacterial, and algal sources, indicates that, based on the distance parameter, the intra-monomer pathway would be favored kinetically. (III) Separation of quinone binding sites. A consequence of the dimer structure and the position of the Q/QH(2) binding sites is that the p-side QH(2) oxidation and n-side Q reduction sites are each well separated. Therefore, in the event of an overlap in residence time by QH(2) or Q molecules at the two oxidation or reduction sites, their spatial separation would result in minimal steric interference between extended Q or QH(2) isoprenoid chains. (IV) Trans-membrane QH(2)/Q transfer. (i) n/p-side QH(2)/Q transfer may be hindered by lipid acyl chains; (ii) the shorter less hindered inter-monomer pathway across the complex would not pass through the center of the cavity, as inferred from the n-side antimycin site on one monomer and the p-side stigmatellin site on the other residing on the same surface of the complex. (V) Narrow p-side portal for QH(2)/Q passage. The [2Fe-2S] cluster that serves as oxidant, and whose histidine ligand serves as a H(+) acceptor in the oxidation of QH(2), is connected to the inter-monomer cavity by a narrow extended portal, which is also occupied in the b(6)f complex by the 20 carbon phytyl chain of the bound chlorophyll., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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 Å 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).

The crystal structure of the cyanobacterial cytochrome b(6)f complex has previously been solved to 3.0-A resolution using the thermophilic Mastigocladus laminosus whose genome has not been sequenced. Several unicellular cyanobacteria, whose genomes have been sequenced and are tractable for mutagenesis, do not yield b(6)f complex in an intact dimeric state with significant electron transport activity. The genome of Nostoc sp. PCC 7120 has been sequenced and is closer phylogenetically to M. laminosus than are unicellular cyanobacteria. The amino acid sequences of the large core subunits and four small peripheral subunits of Nostoc are 88 and 80% identical to those in the M. laminosus b(6)f complex. Purified b(6)f complex from Nostoc has a stable dimeric structure, eight subunits with masses similar to those of M. laminosus, and comparable electron transport activity. The crystal structure of the native b(6)f complex, determined to a resolution of 3.0A (PDB id: 2ZT9), is almost identical to that of M. laminosus. Two unique aspects of the Nostoc complex are: (i) a dominant conformation of heme b(p) that is rotated 180 degrees about the alpha- and gamma-meso carbon axis relative to the orientation in the M. laminosus complex and (ii) acetylation of the Rieske iron-sulfur protein (PetC) at the N terminus, a post-translational modification unprecedented in cyanobacterial membrane and electron transport proteins, and in polypeptides of cytochrome bc complexes from any source. The high spin electronic character of the unique heme c(n) is similar to that previously found in the b(6)f complex from other sources.

The structure and function of the cytochrome b6f 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 b6f 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 c(n), not found in the bc1 complex, but electronically tightly coupled to the heme b(n) of the b6f complex, is included. Crystal structures of the cyanobacterial complex with the quinone analogue inhibitors, NQNO or tridecyl-stigmatellin, show the latter to be ligands of heme c(n), implicating heme c(n) 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 b6f complex and heme c(n) in regulation of the relative rates of noncyclic and cyclic electron transfer. The likely presence of cyclic electron transport in the b6f complex, and of heme c(n) in the firmicute bc complex suggests the concept that hemes b(n)-c(n) 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.