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Purification and Crystallization of the Cytochrome b6f Complex in Oxygenic Photosynthesis

  • Huamin Zhang
  • William A. Cramer
Part of the Methods In Molecular Biology™ book series (MIMB, volume 274)

Abstract

The cytochrome b 6 f complex from the thermophilic cyanobacterium Mastigocladus laminosus and spinach chloroplasts has been purified as a dimeric species. It was found by electrospray ionization mass spectroscopy to contain eight and nine subunits, respectively, and dimeric masses of 217,070 and 286,454 Da. The subunits common to the complex from both sources are petA (cytochrome f), B (cytochrome b 6), C (Rieske iron-sulfur protein), D (subunit IV), and small 3.2–4.2 kDa polypeptides petG,L,M, and N. The ninth polypeptide, the 35 kDa petH polypeptide in the spinach complex, was identified as ferredoxin NADP reductase (FNR), which binds to the complex tightly at a stoichiometry of approx 0.9 (cyt f)−1. The spinach complex contains diaphorase activity diagnostic of FNR, and is active in facilitating ferredoxin-dependent electron transfer from NADPH to the cytochrome b 6 f complex. The purified cytochrome b 6 f complex contains stoichiometrically bound chlorophyll a and β-carotene at a ratio of one per cytochrome f, and bound lipid, in which MGDG and PG are the most abundant species. The delipidated highly purified complexes are active immediately after preparation and for approx 1 wk if left on ice, transferring 300–350 electrons/cyt f/s. Both complexes are subject to proteolysis and associated loss of activity if left for extended periods (>1 wk) at room temperature. Addition of pure synthetic lipid to the delipidated M. laminosus complex (the “lipid augmentation” technique) allows rapid and ready formation of large (>0.2 mm) crystals suitable for x-ray diffraction analysis and structure determination, which diffract with good statistics to 3.0 Å.

Key Words

Cytochrome (cyt) dimethyl sulfoxide (DMSO) ethylenedinitrilo tetraacetic acid (EDTA) ferredoxin:NADP+ oxidoreductase (FNR) monogalactosyl-diacylglycerol (MGDG) phosphatidylglycerol (PG) plastoquinone bound on electrochemically negative side of b6f complex (Qnn-undecyl-β-D-maltoside (UDM) 

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Copyright information

© Humana Press Inc., Totowa, NJ 2004

Authors and Affiliations

  • Huamin Zhang
    • 1
  • William A. Cramer
    • 1
  1. 1.Department of Biological Sciences, Lilly Hall of Life SciencesPurdue UniversityWest Lafayette

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