Abstract
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.
Highlights
It is unexpected that stable intact dimeric b6f complex suitable for crystal growth has far not been obtained from unicellular cyanobacteria, but only from the filamentous moderately thermophilic M. laminosus
This situation motivated a search for a cyanobacterium that could be used in structure and function studies of the b6f complex that would include the potential for mutagenesis
Signal—X-band EPR spectra of b6f complex purified from Nostoc, displayed in Fig. 4 for perpendicular and parallel orientations of the microwave magnetic field, show the very large g value, g ϭ 12, described previously in spectra derived from the M. laminosus b6f complex, which clearly show that hemes bn and cn are spin-coupled
Summary
Materials—Ammonium sulfate was purchased from MP Biomedicals (Solon, OH). Decyl-plastoquinol and propyl-agarose were from Sigma-Aldrich, SP-Sepharose from Amersham Biosciences, and 1,2-dioleoyl-sn-glycero-3-phosphocholine and n-undecyl--D-maltopyranoside from Anatrace (Maumee, OH). Further purification using ammonium sulfate precipitation, hydrophobic chromatography purification, and density centrifugation in sucrose gradient was performed as described [11], except that TNE buffer was used in all steps. Solid ammonium sulfate was added to the supernatant of broken Nostoc cells (in the first step of the thylakoid membrane isolation described above) to achieve 45% saturation and stirred for 2 h at 4 °C. The pellet was resuspended in buffer A (1 mM MES, pH 6.5, 0.25 mM ferricyanide), and ammonium sulfate was removed using a Centriprep YM-10 concentrator (Millipore, Billerica, MA). This sample was loaded onto a SP-Sepharose column equilibrated with buffer A. Crystallization of the b6f Complex—Crystals of the native cytochrome b6f complex were obtained using the modified hanging drop, vapor-diffusion method described before [2]
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