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)


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) 


  1. 1.
    Kallas, T. (1994) The cytochrome b 6 f complex, in The Molecular Biology of Cyanobacteria (Bryant, D. A., ed.), Kluwer Academic Publishers, Dordrecht, pp. 259–317.Google Scholar
  2. 2.
    Cramer, W. A., Soriano, G. M., Ponomarev, M., et al. (1996) Some new structural aspects and old controversies concerning the cytochrome b 6 f complex of oxygenic photosynthesis. Ann. Rev. Plant Physiol. Plant Mol. Biol. 47, 477–508.CrossRefGoogle Scholar
  3. 3.
    Soriano, G. M., Ponamarev, M. V., Carrell, C. J., Xia, D., Smith, J. L., and Cramer, W. A. (1999) Comparison of the cytochrome bc 1 complex with the anticipated structure of the cytochrome b 6 f complex: De plus ca change de plus c’est la meme chose. J. Bioenerg. Biomembr. 31, 201–213.PubMedCrossRefGoogle Scholar
  4. 4.
    Zhang, H., Whitelegge, J. P., and Cramer, W. A. (2001) Ferredoxin:NADP+ oxidoreductase is a subunit of the chloroplast cytochrome b 6 f complex. J. Biol. Chem. 276, 38,159–38,165.PubMedGoogle Scholar
  5. 5.
    Whitelegge, J. P., Zhang, H., Taylor, R., and Cramer, W. A. (2002) Full subunit coverage liquid chromatography electrospray-ionization mass spectrometry (LCMS+) of an oligomeric membrane protein complex: the cytochrome b 6 f complex from spinach and the cyanobacterium, M. laminosus. Mol. Cellular Proteomics 1, 816–827.CrossRefGoogle Scholar
  6. 6.
    Huang, D., Everly, R. M., Cheng, R. H., et al. (1994) Characterization of the chloroplast cytochrome b 6 f complex as a structural and functional dimer. Biochemistry 33, 4401–4409.PubMedCrossRefGoogle Scholar
  7. 7.
    Pierre, Y., Breyton, C., Kramer, D., and Popot, J. L. (1995) Purification and characterization of the cytochrome b 6 f complex from Chlamydomonas reinhardtii. J. Biol. Chem. 270, 29,342–29,349.PubMedCrossRefGoogle Scholar
  8. 8.
    Widger, W. R., Cramer, W. A., Herrmann, R. G., and Trebst, A. (1984) Sequence homology and structural similarity between the b cytochrome of mitochondrial complex III and the chloroplast b 6 f complex: position of the cytochrome b hemes in the membrane. Proc. Natl. Acad. Sci. USA 81, 674–678.PubMedCrossRefGoogle Scholar
  9. 8a.
    Kurisu, G., Zhang, H., Smith, J. L., and Cramer, W. A. (2003) Structure of the cytochrome b 6 f complex of oxygenic photosynthesis: tuning the cavity. Science 302, 1009–1014.PubMedCrossRefGoogle Scholar
  10. 9.
    Vener, A., van Kan, P. J., Rich, P. R., Ohad, I. I., and Andersson, B. (1997) Plastoquinol at the quinol oxidation site of reduced cytochrome bf mediates signal transduction between light and protein phosphorylation: thylakoid protein kinase deactivation by a single-turnover flash. Proc. Natl. Acad. Sci. USA 94, 1585–1590.PubMedCrossRefGoogle Scholar
  11. 10.
    Xia, D., Yu, C.-A., Kim, H., et al. (1997) Crystal structure of the cytochrome bc 1 omplex from bovine heart mitochondria. Science 277, 60–66.PubMedCrossRefGoogle Scholar
  12. 11.
    Zhang, Z., Huang, L., Shulmeister, V. M., et al. (1998) Electron transfer by domain movement in cytochrome bc 1. Nature 392, 677–684.PubMedCrossRefGoogle Scholar
  13. 12.
    Kim, H., Xia, D., Yu, C. A., et al. (1998) Inhibitor binding changes domain mobility in the iron-sulfur protein of the mitochondrial bc 1 complex from bovine heart. Proc. Natl. Acad. Sci. USA 95, 8026–8033.PubMedCrossRefGoogle Scholar
  14. 13.
    Iwata, S., Lee, J. W., Okada, K., et al. (1998) Complete structure of the 11-subunit mitochondrial cytochrome bc1 complex. Science 281, 64–71.PubMedCrossRefGoogle Scholar
  15. 14.
    Hunte, C., Koepke, J., Lange, C., Roßmanith, T., and Michel, H. (2000) Structure at 2.3 Å resolution of the cytochrome bc 1 complex from the yeast Saccharomyces cerevisiae with an antibody Fv fragment. Structure 8, 669–684.PubMedCrossRefGoogle Scholar
  16. 15.
    Lange, C. and Hunte, C. (2002) Crystal structure of the yeast cytochrome bc1 complex with its bound substrate cytochrome c. Proc. Nat. Acad. Sci. USA 99, 2800–2805.PubMedCrossRefGoogle Scholar
  17. 16.
    Berry, E. A., Huang, L.-S., Ban, M., et al. (2002) Crystallographic studies of Rhodobacter capsulatus cytochrome bc 1 complex. Biophys. J. 82, 290a.Google Scholar
  18. 17.
    Martinez, S. E., Huang, D., Szczepaniak, A., Cramer, W. A., and Smith, J. L. (1994) Crystal structure of the chloroplast cytochrome f reveals a novel cytochrome fold and unexpected heme ligation. Structure 2, 95–105.PubMedCrossRefGoogle Scholar
  19. 18.
    Carrell, C. J., Zhang, H., Cramer, W. A., and Smith, J. L. (1997) Biological identity and diversity in photosynthesis and respiration: structure of the lumen-side domain of the chloroplast Rieske protein. Structure 5, 1613–1625.PubMedCrossRefGoogle Scholar
  20. 19.
    Martinez, S., Huang, D., Ponamarev, M., Cramer, W. A., and Smith, J. L. (1996) The heme redox center of chloroplast cytochrome f is linked to a buried five-water chain. Protein Sci. 5, 1081–1092.PubMedCrossRefGoogle Scholar
  21. 20.
    Carrell, C. J., Schlarb, B. G., Bendall, D. S., et al. (1999) Structure of the soluble domain of cytochrome f from the cyanobacterium, Phormidium laminosum. Biochemistry 38, 9590–9599.PubMedCrossRefGoogle Scholar
  22. 21.
    Berry, E. A., Huang, L.-S., Chi, Y., Zhang, Z., Malkin, R., and Fernandez-Velasco, J. G. (1997) The crystallization and structure of a soluble form of Chlamydomonas reinhardtii cytochrome f. Biophys. J. 72, A125.Google Scholar
  23. 22.
    Sainz, G., Carrell, C. J., Ponamarev, M. V., Soriano, G. M., Cramer, W. A., and Smith, J. L. (2000) Interruption of the internal water chain of cytochrome f impairs photosynthetic function. Biochemistry 39, 9164–9173.PubMedCrossRefGoogle Scholar
  24. 23.
    Yan, J. and Cramer, W. A. (2003) Functional insensitivity of the cytochrome b 6 f complex to structure changes in the hinge region of the Rieske iron-sulfur protein. J. Biol. Chem. 278, 20,925–20,933.PubMedCrossRefGoogle Scholar
  25. 24.
    Pierre, Y., Breyton, C., Lemoine, Y., Robert, B., Vernotte, C., and Popot, J.-L. (1997) On the presence and role of a molecule of chlorophyll a in the cytochrome b 6 f complex. J. Biol. Chem. 272, 21,901–21,908.PubMedCrossRefGoogle Scholar
  26. 25.
    Pierre, Y., Chabaud, E., Herve, P., Zito, F., and Popot, J.-L. (2003) Site-directed photochemical coupling of cytochrome b 6 f-associated chlorophyll. Biochemistry 42, 1031–1041.PubMedCrossRefGoogle Scholar
  27. 26.
    Zhang, H., Huang, D., and Cramer, W. A. (1999) Stoichiometrically bound beta-carotene in the cytochrome b 6 f complex of oxygenic photosynthesis protects against oxygen damage. J. Biol. Chem. 274, 1581–1587.PubMedCrossRefGoogle Scholar
  28. 27.
    Metzger, S. U., Cramer, W. A., and Whitmarsh, J. (1997) Critical analysis of the extinction coefficient of chloroplast cytochrome f. Biochim. Biophys. Acta 1319, 233–241.PubMedCrossRefGoogle Scholar
  29. 28.
    Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680–685.CrossRefGoogle Scholar
  30. 29.
    Black, M. T., Widger, W., and Cramer, W. C. (1987) Large-scale purification of active cytochrome b 6 f complex. Arch. Biochem. Biophys. 252, 655–661.PubMedCrossRefGoogle Scholar
  31. 30.
    Sato, N. and Murata, N. (1988) Membrane lipids. Methods Enzymol. 167, 251–259.CrossRefGoogle Scholar
  32. 31.
    Zhang, H., Kurisu, G., Smith, J. L., and Cramer, W. A. (2003) A defined protein-detergent-lipid complex for crystallization of integral membrane proteins: the cytochrome b 6 f complex of oxygenic photosynthesis. Proc. Natl. Acad. Sci. USA 100, 5160–5163.PubMedCrossRefGoogle Scholar

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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