Purification and crystallization of the cyanobacterial cytochrome b6f complex.

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TitlePurification and crystallization of the cyanobacterial cytochrome b6f complex.
Publication TypeJournal Article
Year of Publication2011
AuthorsBaniulis, D, Zhang, H, Zakharova, T, S Hasan, S, Cramer, WA
JournalMethods Mol Biol
Volume684
Pagination65-77
Date Published2011
ISSN1940-6029
KeywordsChemical Fractionation, Chromatography, Crystallization, Cyanobacteria, Cytochrome b6f Complex, Electron Transport, Electrophoresis, Polyacrylamide Gel, Lipids, Mass Spectrometry, Models, Molecular, Nostoc, Pigments, Biological, Protein Conformation, Solubility, Spectrum Analysis, Spinacia oleracea, Sucrose, Thylakoids, Ultracentrifugation
Abstract

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).

DOI10.1007/978-1-60761-925-3_7
Alternate JournalMethods Mol. Biol.
PubMed ID20960122
Grant ListGM-32383 / GM / NIGMS NIH HHS / United States