Purification and crystallization of the cytochrome b6f complex in oxygenic photosynthesis.
The cytochrome b6f 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 b6), 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 poly-peptide 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 b6f complex. The purified cytochrome b6f complex contains stoichiometrically bound chlorophyll a and beta-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 A.
Zhang
Huamin
H
Department of Biological Sciences, Lilly Hall of Life Sciences, Purdue University, West Lafayette, IN, USA.
Cramer
William A
WA
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D003460:Crystallization; D000458:Cyanobacteria / Q000737:chemistry / Q000166:cytology; D045346:Cytochrome b6f Complex / Q000302:isolation & purification* / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D010788:Photosynthesis; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D021122:Protein Subunits / Q000302:isolation & purification / Q000378:metabolism; D018724:Spinacia oleracea / Q000737:chemistry / Q000166:cytology; D020524:Thylakoids / Q000737:chemistry
D001426:Bacterial Proteins; D010940:Plant Proteins; D021122:Protein Subunits; D045346:Cytochrome b6f Complex; D010100:Oxygen