Structure of the cytochrome b subunit of UQH2:cyt c2 oxidoreductase, and the folding of the subunit in the membrane.


This note contains a summary of the structural model developed in the following references:

We have used hydropathy plots, amphipathy analysis, sequence alignment, mean sequence profiles, calculation of conservation moment, and the Walsh-Crofts structural propensity indices, all available in the PSAAM package, to predict a folding pattern for the subunits of the cytochrome b subunit. We were able to suggest a change from the established 9-helix model to one with only 8 transmembrane helices. Subsequent mapping of inhibitor resistant mutants, and characterization of specific mutant strains (in collaboration with Dr. R. Gennis), has lent strong support to the model. We have also use molecular genetics techniques to explore the topology of the cytochrome b subunit through fusion of partial sequences with the phoA gene of alkaline phosphatase. The results strongly support the new 8-helix model. We have used computer model building to suggest a tertiary structure for the cytochrome b subunit(Fig. 1.), showing the location of catalytic sites for oxidation and reduction of quinone, and are using energy minimization and molecular dynamics to generate a plausible physical model.

INTRODUCTION

The ubiquinol:cytochrome c2 oxidoreductase (bc-complex) of Rb. sphaeroides has three main subunits which bear the prosthetic groups, and contribute to three catalytic sites and internal electron transfer pathways which define the modified Q-cycle mechanism. In this note, we report on progress in modelling the structure of cytochrome b of the bc-complex.

MODEL OF CYTOCHROME B.

Fig. 1. shows a preliminary model of the cytochrome b subunit. Several views of the model are presented:

The six transmembrane helices of the model are shown in a view from the cytoplasmic side.

The side view ot the structure shows six transmembrane helices, A-F (out of eight predicted), three amphipathic helices a, ab and cd, the two heme groups of cytochromes (cyt) bH and bL, and two ubiquinone molecules identifying the two quinone reactive catalytic sites. Residues near the putative Qi-site (blue) and Qo-site (orange) discussed below are high-lighted.

A file containing the coordinates of the model in PDB format can be downloaded from this ftp site.

The following constraints were used in building the model:

  • 1) The primary sequence as previously reported [1].
  • 2) Conserved hydrophobic spans which identify putative membrane spanning helices [2-4]. These spans are also identified on the basis of profiles using probability parameters from the distribution of residues in known or predicted membrane helices [5], or in buried helices identified in known structures in the Brookhaven Protein Data Bank [6].
  • 3) Spans showing conserved amphipathy at the helical repeat (hydrophobic moment) [4,7-9]. The identification of helix cd as an amphipathic helix was of importance in suggesting removal of this span from the membrane, to convert the original 9-helix model [2,3] to a structure containing 8 transmembrane helices, A-H (4,9). Amphipathy profiles identify spans at the N-terminal end (a helix), between helices A and B (ab helix), C and D (cd helix), and possibly between E and F (ef helix).
  • 4) A pattern of conservation of residues suggesting a helical motif (mutability moment) [9-12]. The mutability moment shows a vector such that in membrane helices at the lipid-protein interface, the unconserved face is the more hydrophobic face, and faces the lipid. In more amphipathic helices suggested to be at the protein water interface, the conserved face is the more hydrophobic face. This complementary pattern of mutability and hydrophobic moment provides a strong indication of the orientation of these helices at the protein-lipid or protein-water interface. Helices A and ab show strong mutabilty moments, and several of the membrane helices show strong moments for both hydrophobicity and mutability at their ends.
  • 5) The topological organization with eight transmembrane spans has been strongly supported by our phoA fusion experiments [13].
  • 6) Four conserved histidines, two each in helices B and D, which are spaced 14 residues apart so as to fall on the same side of the helix, have been suggested as likely ligands for the two heme groups [2,3]. Site-directed mutagenesis of these residues has demonstrated which histidines ligate which hemes [14].
  • 7) Four conserved glycines, 2 each in helices A and C, spaced, like the liganding histidines, 14 residues apart, are suggested to accommodate the packing needs of the hemes [15]. In support of this suggestion, in helix A, which shows a strong mutability moment, and in helix C, the glycines are on the conserved face of the helix (Fig. 1, 2).
  • 8) Inhibitor resistance mutations. An important feature leading to support of the 8-helix model was the mapping of lesions giving rise to resistance to inhibitors at the two quinone processing catalytic sites. In reaction centers from several bacteria, and photosystem II of green plants and algae, mutations giving rise to resistance to herbicides have all mapped to the QB-site (where quinone is reduced), which is blocked by these inhibitors. As we have previously pointed out [4], this pattern could be used to infer that similar mutations in cyt b would identify residues which contribute to the catalytic sites at which the affected inhibitors bind. In the 8-helix model, residue changes conferring resistance to inhibitors (diuron, antimycin, HQNO) acting at the Qi- (quinone reducing) site all fall on the N- (protochemically Negative) side identified by the location of the heme of cytochrome bH, while those giving resistance to inhibitors (stigmatellin, myxothiazol, mucidin) acting at the Qo- (quinol oxidizing) site fall on the P-side (16-20), close to cyt bL.

  • 9) Specific mutagenesis. Work on mutagenesis of residues thought to contribute to the Qo-site in Rb. capsulatus by Daldal's group (20), and characterization of these mutant strains by Robertson, Dutton and colleagues (21) has been of importance in defining this site. Our own work exploring both Qo- and Qi-sites in Rb. sphaeroides is summarized elsewhere [14,22-26].
  • Degli Esposti et al. have written a comprehensive review of work on structural prediction of cytochrome b, and other aspects of the structure and function of the bc1-complex (32).

    A brief summary of experimental data on the tertiary structure of the bc1-complex can be found here.

    REFERENCES

    1. Yun, C.-H., Beci, R., Crofts, A. R., Kaplan, S. and Gennis, R. B. (1990) Eur. J. Biochem. 194, 399-411.
    2. Widger, W.R., Cramer, W.A., Herrmann, R.G. and Trebst, A. (1984) Proc. Natl. Acad. Sci. 81, 674-678.
    3. Saraste, M. (1984) FEBS Lett. 166, 367-372.
    4. Crofts, A.R., Robinson, H.H., Andrews, K., Van Doren, S. and Berry, E. (1987) In Cytochrome Systems: Molecular Biology and Bioenergetics (Papa, S., Chance, B. and Ernster, L., eds.) pp. 617-624, Plenum Publ., New York.
    5. Rao, J.K. and Argos, P. (1986) Biochim. Biophys. Acta, 869, 197-214.
    6. Walsh, L.L., Bobak, M. and Crofts, A.R. (1990) 4th. Symp. Protein Soc. Abstract.
    7. Eisenberg, D. (1984) Ann. Rev. Biochem. 53, 595-523
    8. Cornette, J.L., Cease, K.B., Margalit, H., Spouge, J.L., Berzofsky, J.A. and DeLisi, C. (1987) J. Mol. Biol. 195, 659-685
    9. Crofts, A.R., Wang, Z., Chen, Y., Mahalingham, S., Yun, C.-H. and Gennis, R.B. (1990) in Highlights in Ubiquinone Research (Lenaz, G., Barnabei, O, Rabbi, A. and Battino, M., eds.) pp. 98-103, Taylor & Francis, London, New York, Philadelphia.
    10. Komiya, H., Yeates, T.O., Rees, D.C., Allen, J.P. and Feher, G. (1988) Proc. Natl. Acad. Sci. USA, 85, 9012-9016
    11. Rees, D.C., DeAntonio, L. and Eisenberg, D. (1989) Science, 245, 510-513
    12. Crofts, A.R., Yun, C.-H., Gennis, R.B. and Mahalingham, S. (1989) in Proceedings of the VIIIth. Internatl. Cong. Photosynth., Stockholm, in Press.
    13. Yun, C.-H., Van Doren, S.R., Crofts, A.R., and Gennis, R.B. (1991) J. Biol. Chem. 266, 10967-10973.
    14. Yun, C.-H., Crofts, A.R. and Gennis, R.B. (1991) Biochemistry. In press.
    15. Tron, T., Crimi, M., Colson, A.-M. and Degli Esposti, M. (1991) Eur. J. Biochem. 199, 753-760.,
    16. di Rago, J.-P., Perea, X. and Colson, A.-M. (1986) FEBS Lett., 208, 208-210.
    17. Colson, A.-M., Meunier, B. and di Rago, J.-P. (1987) in Cytochrome Systems (Papa, S., Chance, B. and Ernster, L., Eds.), pp. 135-136, Plenum Publishing Corp.
    18. Howell, N. & Gilbert, K. (1988) J. Mol. Biol. 203, 607-618.
    19. di Rago, J. P. & Colson, A.-M. (1988) J. Biol. Chem. 263, 12564-12570.
    20. Daldal, F., Tokito, M.K. and Davidson, E. (1989) The EMBO J. 8, 3951-3964
    21. Robertson, D.E., Daldal, F. and Dutton, PL. (1990) Biophys. J. 58, 11249-11260
    22. Yun, C.-H., Wang, Z., Crofts, A.R. and Gennis, R.B. (1992) Examination of the functional roles of five highly conserved residues in the cytochrome b subunit of the bc1 complex of Rhodobacter sphaeroides. J. Biol. Chem. 267, 5901-5909
    23. Van Doren, S.R., Gennis, R.B., Barquera, B. and Crofts, A.R. (1993) Site-Directed Mutations of Conserved Residues of the Rieske Iron-Sulfur Subunit of the Cytochrome bc1 Complex of Rhodobacter sphaeroides Blocking or Impairing Quinol Oxidation. Biochemistry, 32, 8083-8091.
    24. Hacker, B., Barquera, B., Crofts, A.R. and Gennis, R.B. (1993) Characterization of mutations in the cytochrome b subunit of the bc1 complex of Rhodobacter sphaeroides that affect the quinone reductase site (Qc). Biochemistry 32, 4403-4410
    25. Hacker, B., Barquera, B. Gennis, R.B. and Crofts A.R. (1994) Site-Directed Mutagenesis of Arginine-114 and Tryptophan-129 in the Cytochrome b Subunit of the bc1 Complex of Rhodobacter sphaeroides : Two Highly Conserved Residues Predicted to be Near the Cytoplasmic Surface of Putative Transmembrane Helices B and C. Biochemistry, 33, 13022-13031
    26. Crofts, A.R., Barquera, B., Bechmann, G., Guergova, M, Salcedo-Hernandez, R., Hacker, B., Hong, S. and Gennis, R.B. (1996) Structure and function in the bc1-complex of Rb. sphaeroides. Photosynthesis: from light to biosphere. (Mathis, P., ed.), Vol. II, pp. 493-500. Kluwer Academic Publ., Dordrecht.
    27. Xia, Y., Deisenhofer, J., Yu, C.-A., Xia, L. and Yu, L. (1996) Abstracts. Biophys. Soc. Meeting, Baltimore, Feb. 1996.
    28. Xia, D., Kim, H., Deisenhofer, J., Yu, C.-A., Xia, J.-Z. and Yu, L. (1996) Crystal structure of beef heart mitochondrial bc1-complex. Abstracts, #SO355. Intntl. Union Crystallography Meeting, Seattle.
    29. Kim, H., Xia, D., Deisenhofer, J., Yu, C.-A., Kachurin, A. and Yu, L. (1996) Abstracts, #SO356. Intntl. Union Crystallography Meeting, Seattle.
    30. Weiss, H. (1987) Structure of mitochondrial ubiquinol-cytochrome c reductase (Complex III). Curr. Topics in Bioenergetics, 15, 67-90
    31. Karlsson, B., Hovmoller, S., Weiss, H. and Leonard, K. (1983) Structural studies of cytochrome reductase. Subunit topography determined by electron microscopy of membrane crystals of a subcomplex. J. Mol. Biol. 165, 287-302
    32. Degli Esposti, M., De Vries, S., Crimi, M., Ghelli, A., Patarnello, T., and Meyer, A. (1993) Mitochondrial cytochrome b: Evolution and structure of the protein. Biochim. Biophys. Acta 1143, 24-271