Department of  Molecular Membrane Biology Max-von-Laue-Str. 3, D-60438 Frankfurt am Main, Germany  
 
C. Roy D. Lancaster
 
Structural and Mechanistic Membrane Biochemistry

The Group has moved to Saarland University.
These pages are no longer updated.

 
Dr. phil. nat., University of Frankfurt, Germany, 1996
Habilitation (Biochemistry), University of Frankfurt, 2003
Group Leader since 2000
Tel.: +49 69 6303 1013
Fax: +49 69 6303 1002
E-mail
Last update:Monday, 11-May-2009 18:01:01 CEST
* GROUP MEMBERS
* SELECTED PUBLICATIONS
* DIPLOMA THESES
* DOCTORAL THESES
* GRANT SUPPORT
* AWARD
* INVITED LECTURES
* TEACHING
 
PREVIOUS AND CURRENT  RESEARCH
 
We are studying the mechanisms of action of selected membrane protein complexes on the basis of accurately determined structures. The first of the two most advanced projects is on the quinol: fumarate reductase (QFR) from the anaerobic bacterium Wolinella succinogenes, where fumarate reduction to succinate is coupled to the oxidation of menaquinol to menaquinone (cf. Fig. 1). This enzyme is similar in composition and function - and also in structure - to succinate dehydrogenase, which is complex II of the aerobic respiratory chain. Succinate dehydrogenases and fumarate reductases are collectively referred to as succinate: quinone oxidoreductases (SQORs). 
 
Fig. 1. Electron transport system of Wolinella succinogenes (click on image to enlarge).
 
The second major project concerns the photosynthetic reaction centre from the non-sulfur purple bacterium Rhodopseudomonas (Rp.) viridis, where light-induced electron transfer and coupled proton transfer reactions result in the reduction of ubiquinone to ubiquinol at the binding site of the secondary electron acceptor quinone QB (cf. Fig. 2).
 
Fig. 2. Photophosphorylation in Rp. viridis. The QB site in the reaction centre is highlighted in white (click on image to enlarge).

Our work involves site-directed mutagenesis, membrane protein production, purification, functional characterization, and crystallization, as well as X-ray diffraction analysis, crystallographic refinement and theoretical analysis of the resulting atomic models. In the case of the photosynthetic reaction centre, the implications for the coupling of electron and proton transfer of the structure of a reaction centre with a ubiquinone bound stoichiometrically to the QB site have been analysed theoretically by continuum electrostatics. Furthermore, we have derived a mechanistic model for QB reduction and protonation by determining structures of reaction centres modified at the QB site. X-ray structure analysis of other modified Rp. viridis reaction centres has been performed up to a resolution of 2.0 Å (Lancaster et al. (2000) J. Biol. Chem. 275, 39364-39368).

In the case of W. succinogenes QFR, we have solved its three-dimensional structure by X-ray crystallography at 2.2 Å resolution (Lancaster et al. (1999) Nature 402, 377-385) [see press release]. The structure of the three protein subunits A, B, and C and the arrangement of the six prosthetic groups (a covalently-bound FAD, three iron-sulfur clusters, and two haem b groups; cf. Fig. 3) suggests a pathway of electron transfer from the quinol-oxidising dihaem cytochrome b in the membrane to the site of fumarate reduction in the hydrophilic subunit A.
 
Fig. 3. Three-dimensional structure of the W. succinogenes QFR dimer of heterotrimeric complexes of A (blue and blue-green), B (red and purple), and C (green and light blue) subunits. From top to bottom, the six prosthetic groups per heterotrimer are the covalently bound FAD, the [2Fe-2S], the [4Fe-4S], and the [3Fe-4S] iron-sulfur clusters, the proximal and the distal heme b groups.
 
Based on crystallographic analysis of three different crystal forms of the enzyme and the results from site-directed mutagenesis, we have derived a mechanism of fumarate reduction and succinate oxidation,  which is generally applicable throughout the superfamily of succinate:quinone oxidoreductases (Lancaster et al. (2001) Eur. J. Biochem. 268, 1820-1827). By combining the results from site-directed mutagenesis, functional and electrochemical characterisation, and X-ray crystallography, we have identified a residue which is essential for menaquinol oxidation. The location of this residue in the structure indicates that the coupling of the oxidation of menaquinol to the reduction of fumarate by W. succinogenes QFR should be associated with the generation of a transmembrane electrochemical potential (Fig. 4a). The latter could not be confirmed. A hypothesis has been presented which reconciles these apparently conflicting experimental observations (Fig. 4b). First experiments supporting this hypothesis have been performed.
 
Fig. 4. The coupling of electron and proton flow in W. succinogenes QFR. The positive side of the membrane is the periplasm, the negative side the cytoplasm.
a)  Hypothetical transmembrane electrochemical potential as suggested by the arrangement of the catalytic sites of fumarate reduction and menaquinol oxidation in the structure (Lancaster et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 13051-13056).
b) Hypothetical cotransfer of one H+ per electron across the membrane („E-pathway hypothesis“; Lancaster (2002) Biochim. Biophys. Acta 1565, 215-231). The two protons that are liberated upon oxidation of menaquinol (MKH2) are released to the periplasm via the residue Glu C66. In compensation, coupled to electron transfer via the two heme groups, protons are transferred from the periplasm via the ring C propionate of the distal heme bD and the residue Glu C180 to the cytoplasm, where they replace those protons which are bound during fumarate reduction. In the oxidized state of the enzyme, the “E-pathway” is blocked.
 
FUTURE  PROJECTS
 
To understand the structure-function relationships of RCs and SQORs by determining the three-dimensional structures of modified Rp. viridis RCs and of W. succinogenes QFRs as well as of SQORs from other species, and by performing theoretical calculations on the obtained atomic models. Possibly, to extend this to other bioenergetically relevant quinone-binding and/or dihaem-containing membrane protein complexes.

 
Group Photo September 2006
Group Photo September 2006 (from left to right): 
Roy Lancaster, Nicole Hilgendorff, Rajsekhar Paul, Lucia Cenacchi, Florian Müller, Elena Herzog, Hanno Juhnke.


GROUP MEMBERS
  • Technical assistant:
    • Imke Wüllenweber (e-mail)
  • Post-doctoral associate:
    • Dr. Hanno Juhnke (e-mail)
  • Masters student:
    • Manuela Busch (e-mail)
SELECTED PUBLICATIONS
  • Madej, M.G., Nasiri, H.R., Hilgendorff, N.S., Schwalbe H., and Lancaster, C.R.D.: Evidence for transmembrane proton transfer in a dihaem-containing membrane protein complex. EMBO J. 25, 4963-4970 (2006), doi: 10.1038/sj.emboj.7601361.
    •


SELECTED DIPLOMA THESES
  • Philipp Schleidt: Charakterisierung von durch gezielten Aminosäureaustausch generierten Varianten der Chinol:Fumarat-Reduktase aus Wolinella succinogenes. Frankfurt 2007.
    •
  • Florian Müller: Produktion der C4-Dicarboxylat-Transporter DcuA, DcuB, DcuC und "Transporter" aus Salmonella typhimurium in Escherichia coli. Frankfurt 2006.
  • Heiko Hiltscher: Generierung durch gezielten Aminosäureaustausch von Varianten der Succinat-Dehydrogenase aus Wolinella succinogenes und deren Charakterisierung. Frankfurt 2005.
DOCTORAL THESES
  • Lucia Cenacchi: Heterologous Production and Characterization of Two Distinct Di-Heme Containing Membrane Integral Cytochrome b561 Enzymes from Arabidopsis thaliana. Frankfurt 2007.
    •
  • Hanno Juhnke: Molekularbiologische und biochemische Charakterisierung der "Succinat-Dehydrogenase" aus Wolinella succinogenes. Frankfurt 2007.
  • Mauro Mileni: Biochemical, Structural and Functional Characterization of Diheme-Containing Quinol:Fumarate Reductases: the Role of Heme Propionates and the Enzymes from Pathogenic Epsilon-Proteobacteria. Frankfurt 2005.
  • Alexander H. Haas: Investigation of Coupled Electron and Proton Transfer in the Quinol: Fumarate Reductase from Wolinella succinogenes with Electrochemically Induced FTIR and VIS Difference Spectroscopy and Multiconformation Continuum Electrostatic Calculations. Frankfurt 2004.
SELECTED INVITED LECTURES
  •  [Full list 1997-2005]
  •    MRC Dunn Human Nutrition Unit (Cambridge, UK, 14 Feb 2007)
  •    8th International Conference on Membrane Redox Systems and Their Role in Biological Stress and Disease (Szeged, Hungary, 4-8 Apr 2006)
  •    International Meeting on "Microbial Respiratory Chains" (Tomar, Portugal, 19-23 Mar 2006)
  •    Annual Meeting of the German Biophysical Society (Freiburg, Germany, 13 Sep 2004)
  •    Gordon Research Conference on Molecular and Cellular Bioenergetics (Proctor Academy, Andover, NH, U.S.A, 20 Jun 2004)
  •    Nobel Symposium on “Membrane Proteins: Structure, Function and Assembly” (Stockholm, Sweden, 22 Aug 2003)
  •   Gordon Research Conference on Biophysical Aspects of Photosynthesis (Roger Williams University, NH, U.S.A, 25 Jun 2003)
  •  Gordon Research Conference on Protons & Membrane Reactions (Ventura, CA, U.S.A., 26 Feb 2003) 
  •   XIX International Union of Crystallography (IUCr) Congress, Symposium on "Structure and Function of Membrane Proteins" (Geneva, Switzerland, 13 Aug 2002) 
  •   International Conference on "Sequence, Structure and Function in Membrane Protein Systems" (Zichron Ya'acov, Israel, 6 Nov 2001) 
  •   International Conference on the Structure, Dynamics and Function of Proteins in Biological Membranes (Monte Verita, Ascona, Switzerland, 17 Mar 2001) 

    •  
GRANT SUPPORT


AWARD
  •     Boris Rajewsky Prize in Biophysics (2007)
TEACHING
  •  SS 2007   Advanced Lab Course: Biochemistry 
  • IMPResS 06: Lecture Course on Structure and Function of Biological Membranes (plus a one-week Workshop)
  •  WS 07/08  Lectures & Seminars: Biophysical Chemistry IV: Spectroscopy and X-Ray Diffraction (plus an introductory Practical Course) 
 
   

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