Over the course of evolution, the endosymbiont was transformed into the chloroplast, an organelle which still possesses its own genome and its own gene-expression machinery. Presence of the lipids in the structure is not the only factor required to ensure dimer stability, since the dimeric b6f complex is retained prior to lipid augmentation. Yuan Li, Gary J. Loake, in Plant Transcription Factors, 2016. CES is not restricted to the integral thylakoid membrane proteins: the large subunit of Rubisco is also a CES protein, whose rate of synthesis is highly reduced in deletion strains lacking expression of the small subunit (Khrebtukova and Spreitzer, 1996). Conversely, its absence does not affect the rate of synthesis of other PS II subunits, even though they are more rapidly degraded in the absence of CP43 (de Vitry et al., 1989). The light-dependent reaction takes place within specialized membrane discs of the chloroplast, known as thylakoid. Refinement of the model was carried out with a 3.0 Å data set from a second crystal with the quinone-analog inhibitor tridecyl-stigmatellin. Although neither of the photosystems directly moves protons across the membrane they are important examples of proteins which principally contribute to the generation of the proton electrochemical gradient as a result of moving electrons across the membrane. It is a unique Fe-2S] cluster in that one of the two Fe atoms is coordinated by two histidine residues rather than … 1). For this purpose, the precipitated RNA is resuspended in formamide dye [deionized formamide, 0.1%(w/v) xylene cyanol, 0.1 %(w/v) bromphenol blue], heated to 85° for 2 min, and loaded onto a denaturing acrylamide gel (minigel) [5%(w/v) acrylamide (19:1), 8 M urea in 0.5× TBE], Gels are preelectrophoresed for 1 hr at 200 V in 0.5× TBE before sample loading. The function of the chlorophyll, is unknown, but it may fill structure gaps, similarly to bound lipids in membrane proteins. The cavity floor is formed by the N-terminal 25 residues of cytochrome b6 and by lipid head groups that fill the cavity in situ. The binding of a second plastoquinol causes one electron to reduce oxidized plastocyanin in the high-potential electron transport chain. The full-length transcript (300 nucleotides) is excised and eluted from the gel matrix in an Eppendorf tube by adding 180 µl of diethyl pyrocarbonate (DEPC)-treated water, 20 µl of DEPC-treated 3 M sodium acetate (pH 5.5), 1 µl of DEPC-treated 0.25 M EDTA, and 1 µl of 20% (w/v) sodium dodecyl sulphate (SDS). Cytochrome b6f and cytochrome bc1 carry out similar electron transport functions despite being located in the chloroplast and mitochondria respectively. The coupling of the oxidation-reduction and protonation-deprotonation is central to the mechanism of proton translocation in the complex (2). Three lipid molecules, a sulfo-lipid and two DOPC molecules, can be resolved in the intermonomer quinone exchange cavity. In this study, the Q104C mutant cytochrome f protein was spin labeled by a nitroxide radical at position 104, and the dipole interaction between the spin label and the intrinsic low-spin ferric heme center was studied. Cytochrome f from the cytochrome b6f complex is a typical CES protein whose rate of synthesis decreases in the absence of cytochrome b6 or subunit IV, to about 10% of that observed in the wild type (Lemaire et al., 1986; Kuras and Wollman, 1994). The in vivo experiments may contain some factor(s) that are missing in the in vitro system that alter the energetic requirements of the transport reaction (Theg et al., 2005). The unicellular alga Chlamydomonas reinhardtii belongs to the group of eukaryotic green algae and lives in freshwater or in moist places. The complex functions as a dimer. This interpretation invokes an entropic role for the ΔpH, consistent with indications that a ΔpH is required for the assembly of the Tat translocase in isolated thylakoids (Mori and Cline, 2002). The dipolar contribution was separated by dividing the RIDME traces recorded at different TR(2) values by that with TR1=5μs, and mostly manifested itself as a “bump” at t = 0 and with a half-with of ~ 15 ns. 72 • The electrons from cytochrome b 6 f are passed to another mobile electron carrier, a water-soluble, copper-containing, peripheral membrane protein called plastocyanin, situated on the luminal side of the thylakoid membrane • Plastocyanin carries electrons to the luminal side of PSI, where they are transferred to P700 +, the positively charged reaction-center pigment of PSI. The xanthophylls lutein and violaxanthin are bound to the light-harvesting antennae of both photosystems, while loroxanthin and neoxanthin appear to be confined to the light-harvesting complexes of PS II (Pineau et al., 2001). Photosynthesis is the basis of life on Earth, which provides the food, oxygen and energy that sustain the biosphere and human civilization. In this view, both CP47 and D1 are CES subunits in PS II biogenesis. The starting point for all biological solar-driven H2-production methods is the process of photosynthesis (see Figure 1). The tight openings of the p-side and n-side cavities allow enough space for plastoquinol and plastoquinone entry. Three prosthetic groups in the b6f complex, the heme cn, one chlorophyll a and one β-carotene molecule per b6f monomer, are uniquely found in the b6f complex, but not in the bc1 complex (3). Schematic representation of the major photosynthetic proteins. The two monomers form a protein-free central cavity on each side of the thylakoid membrane for the p-side and n-side domains. The coupling of the oxidation-reduction and protonation-deprotonation is central to the mechanism of proton translocation in the complex (2). The electron transfer from heme bp to heme b, on the stroma side of the complex allows heme b, to reduce plastoquinone-9 to a semiquinone. However, a NOS with significant sequence similarity to human eNOS has been found in the unicellular marine green algae, Ostreococcus tauri (Foresi et al., 2010). Photosynthesis extracts electron from H2O and via the electron transport chain delivers them to the [FeFe]-hydrogenase where H2 is generated. Murthy, M.L. The three-dimensional structures of the p-side, n-side, and transmembrane domains facilitate their roles in enzyme activity. An obstacle in the crystal structure studies has been proteolysis and resulting monomerization of the complex, which degrades the complex from its active dimer form. Each monomer consists of four large subunits, including cytochrome f, cytochrome b6, the Rieske iron-sulfur protein (ISP), and subunit IV; as well as four small hydrophobic subunits, PetG, PetL, PetM, and PetN. With respect to subcellular distribution, the carotenoids in vegetative cells localize to the chloroplast where they either serve as photosynthetic pigments bound to the protein complexes of the two photosystems (see Chapter 14), or as components of the eyespot apparatus (see Volume 3, Chapter 13). Oxygenic photosynthesis originated more than three billion years ago when primordial cyanobacteria evolved an apparatus capable of utilizing sunlight for the extraction of electrons from water (Crowe et al., 2013). Cytochrome B6f Complex Organellar and Metabolic Processes. Photosynthetic reducing power can thus be partitioned between at least two pathways: CO2 reduction (under aerobic conditions) and H2 production (under anaerobiosis). Cys35 on the n side of the A helix makes a single covalent thioether bond with heme x, placing the prosthetic group in close proximity to the center of the complex. Cyclooxygenase 2, an enzyme involved in inflammation, is a cytochrome b protein. Cytochrome b6f (1VF5) from Mastigocladus laminosus is an integral membrane protein that mediates electron transfer between the photosystem II and photosystem I reaction centers by oxidizing lipophilic plastoquinol and reducing plastocyanin. In PSII, electron transfer may lead to formation of a singlet oxygen (1O2) at the stroma. Initially heterotrophic eukaryotes first acquired the ability to perform oxygenic photosynthesis by entering into an endosymbiotic relationship with an ancestral cyanobacterium (Gray and Archibald, 2012), and modern algae and plants descended from such organisms. Apocytochrome f is a mutant version of cytochrome f with a 6 His tag at the C-terminus. In mammals, three isoforms of NO synthase (NOS) have been characterized (nNOS, eNOS, and iNOS; Alderton et al., 2001). The results of DALI (Z=26.4) and protein BLAST (E=7e-135) searches show that the cyt b6f of C. reinhardtii has greater primary and tertiary similarities with the cyt b6f of M. laminosus than were found with cytochrome bc1. The structure was solved by isomorphous replacement using Pb and Pt derivatives and multiwavelength anomalous diffraction from native iron atoms (1). In linear electron transfer, an electron extracted from water in the thylakoid lumen is transferred through photosystem II (PSII), plastoquinone (PQ), cytochrome b6f (Cyt), plastocyanin (PC), and photosystem I (PSI) into the chloroplast stroma. The 4 large subunits of the cytochrome b6-f complex are cytochrome b6, subunit IV (17 kDa polypeptide, PetD), cytochrome f and the Rieske protein, while the 4 small subunits are PetG, PetL, PetM and PetN. In addition to photosynthesis, unicellular green algae are able to perform many biotechnologically interesting metabolic reactions, such as fermentation and hydrogen photoproduction. Electrons are shuttled between the two photosystems via the cytochrome b6f (Cytb6f) complex by two mobile, low-molecular-weight carriers–plastoquinone and plastocyanin–while a proton gradient is generated across the membrane to drive an ATP synthase (see Fig. Instead, the latter is downregulated in the absence of ATP production (see section H2 Production: Issues and Challenges). The positive surface potential of cytochrome b, on the stromal side would facilitate docking of anionic ferredoxin to the n side of the complex near heme x. Heme c, acts a redox cofactor on cytochrome f and provides a binding site for tridecyl-stigmatellin, a p-side quinone analog inhibitor. The Rieske iron-sulfur protein suffered cleavage of a, f and subunit IV polypeptides were also clipped at their exposed termini. The PetL subunit of cytochrome b6f is not essential for biogenesis and function, but removal in plant cells affected the mature leaves of tobacco plants. One of these sites in the b6f complex is occupied by a natural sulfo-lipid which interacts with n-side segments of the ISP and cytochrome f (3). The membrane-embedded CF0 portion of the ATP synthase is a selective proton channel that comprises subunits I, II, III, and IV, assembled in a 1:1:(13–14):1 stoichiometry (Seelert et al., 2003; Meyer zu Tittingdorf et al., 2004). The reduced iron-sulfur protein transfers an electron through cytochrome f to plastocyanin. Concomitantly, protons (H+) are carried from the stromal to the lumenal side of the thylakoids, creating a proton gradient across the thylakoid membrane, which is utilized by the enzyme adenosine triphosphate (ATP) synthase to generate the ATP that is also required for CO2 fixation. The hemes are: Heme x, Heme b n, and Heme b p, all bound by cytochrome b 6; Heme f is sequestered by cytochrome f. The correct processing at the 3′ end of the petD mRNA can be reproduced in vitro with a petD 3′-UTR RNA probe synthesized by transcription from a plasmid carrying the petD 3′ UTR.6 The synthetic RNA contains 70 nucleotides of the coding region and extends 58 nucleotides 3′ proximal to the stem–loop, which itself is 46 nucleotides long. Cytochrome b, f generates a transmembrane electrochemical proton gradient for adenosine triphosphate synthesis in the thylakoid membrane of chloroplasts in plants, cyanobacteria, and green algae. The transmembrane region between the b ( 6 ) f and bc ( )! Has not been found in bacteria and the cytochrome b6f has two primary functions: the oxidation of quinols the! A single covalent thioether bond at a position between heme b, minor. In size and the H2-evolution processes eicosane is necessary to purify the full-length transcript gel., site of the RNA template is then resuspended in 100 µl of DEPC-treated water and stored in aliquots! 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