Purification and Crystallization of the Cyanobacterial Cytochrome b 6 f Complex (original) (raw)
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Journal of Biological Chemistry, 2009
The crystal structure of the cyanobacterial cytochrome b 6 f complex has previously been solved to 3.0-Å 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 3.0 Å (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°a bout the ␣and ␥-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 9 Å projection structure of cytochrome b6f complex determined by electron crystallography
Journal of Molecular Biology, 1999
Thin three-dimensional crystals of the cytochrome b 6 f complex from the unicellular algae Chlamydomonas reinhardtii have been grown by Bio-Beads-mediated detergent removal from a mixture of protein and lipid solubilized in Hecameg. Frozen-hydrated crystals, exhibiting p22 1 2 1 plane group symmetry, were studied by electron crystallography and a projection map at 9 A Ê resolution was calculated. The crystals (unit cell dimensions of a 173.5 A Ê , b 70.0 A Ê and g 90.0) showed the presence of dimers, and within each monomer 14 domains of electron density were observed. The combination of the projection map obtained from iceembedded crystals of cytochrome b 6 f with a previous map obtained from negatively stained samples brings new insight in the organization of the complex. For example, it distinguishes some peaks and/or domains that are only extramembrane or transmembrane, and reveals the possible localization of single-stranded transmembrane a-helices (Pet subunits). Furthermore, the cross-correlation of our projection map from frozen hydrated samples with the atomic model of the transmembrane part of the cytochrome bc 1 complex has allowed us to localize the cytochrome b 6 at the dimer interface and to reveal structural differences between the two complexes.
FEBS Letters, 1997
Fractionation of photosynthetic membranes from the cyanobacterium Synechocystis 6803 by polyacrylamide gel electrophoresis in the presence of Deriphat-160 allowed the isolation of a number of pigmented bands. Two of them, with molecular masses of 240 ±20 and 110 ±15 kDa respectively, showed peroxidase activity and, by means of polypeptide composition, immunoblotting and N-terminal sequencing, were identified as dimeric and monomeric cytochrome b¿f complexes, containing 1.3±0.35 chlorophyll molecules per cytochrome/ Further fractionation of monomeric complexes by mild gel electrophoresis in the presence of sodium dodecyl sulfate indicated that it is the cytochrome b 6 polypeptide which provides the actual binding site for the chlorophyll molecule observed in the complex.
Characterization of the Chloroplast Cytochrome b6f Complex as a Structural and Functional Dimer
Biochemistry, 1994
Size analysis of the cytochrome b 6 f complex by FPLC Superose-12 chromatography and Blue Native PAGE indicated a predominantly dimeric component with M r = (1.9-2.5) × 10 5. The true dimer molecular weight including bound lipid, but not detergent, was estimated to be 2.3 × 10 5. Size and shape analysis by negative-stain single-particle electron microscopy indicated that the preparation of dimeric complexes contains a major population that has a protein cross section 40% larger than the monomer, binds more negative stain, and has a geometry with a distinct 2-fold axis of symmetry compared to the monomeric complex. The dimeric species is more stable at higher ionic strength with respect to conversion to the monomeric species. SDS-PAGE of monomer and dimer preparations indicated that both contain the four major polypeptides in approximately equal stoichiometry and also contain the petG M r 4000 subunit. One bound chlorophyll a per monomer, part of the bound lipid, is present in monomer and dimer. The in vitro electron-transport activity (decyl-PQH 2 →PC-ferricyanide) of the separated dimer was comparable to that of the isolated b 6 f complex and was 4-5-fold greater than that of the monomer preparation, whose activity could be attributed to residual dimer. No difference in the properties of the dimer and monomer was detected by SDS-PAGE or redox difference spectrophotometry that could account for the difference in activities. However, the concentration of the Rieske [2Fe-2S] center was found by EPR analysis of the g y = 1.90 signal to be lower in the monomer fraction by a factor of 3.5 relative to the dimer. The presence of active dimer at high levels in the detergent-extracted b 6 f complex, the absence of activity in the monomer, and the absence of a monomer preparation that is not degraded in its spectral properties and activity suggest that the simplest inference is that the dimer is the active complex in the membrane. The possibility that cytochrome b 6 f and bc 1 are primitive trans-membrane-signaling complexes is noted.
Photosynthesis Research, 2004
The recognition that, in photosynthesis, the plastoquinol oxidizing cytochrome b 6f complex resembles the ubiquinol oxidizing cytochrome bc1 complex in respiration is one of the examples of exciting universalization in biological research. A peripheral observation towards the end of 1979 initiated an intensive investigation, which is still ongoing today: next to the ATP synthase the cytochrome b 6f complex could be selectively solubilized from the chloroplast membrane by the combined action of octyl glucoside and cholate. It was mere luck that the isolate was substantially active as an electrogenic, proton translocating plastoquinol—plastocyanin oxidoreductase, and that it also catalyzed oxidant-induced reduction of cytochrome b 6, a signature of the Q-cycle mechanism. The basic findings during the first characterization of the complex are summarized, and the excitement among the collaborating groups is remembered. More recent developments, including the impact of gene technology and the elucidation by the crystal structure, are additionally traced here.