The structure of metalloproteins in solution:

Metalloproteins are ubiquitous electron transfer proteins involved in fundamental biological processes such as photosynthesis, oxidative phosphorylation and nitrogen fixation (Sticht et al., 1995; Sticht & Rösch, 1998; Sticht, 1999). By determining the structure of ferredoxins and rubredoxins containing [4Fe-4S]- [2Fe-2S]- or [1Fe] clusters, respectively, we expect to gain a better understanding of thermostability, halophilism, and the evolution of metalloproteins.

One project concentrates on the cytochrome c6 and the [2Fe-2S] ferredoxin from the thermophilic cyanobacterium Synechococcus elongatus which function as a electron donor and an electron acceptor for photosystem I (PSI), respectively (figure). The structure determination of the cytochrome (Beissinger et al., 1998) and the ferredoxin (Baumann et al., 1996) allowed the identification of those patches of the protein surface that are important for interaction with photosystem I. As PSI from the same organism is known at high resolution, Synechococcus elongatus appears very well suited to investigate photosynthetic electron transfer in detail, and the structure of cytochome c6 provides the basis for mutagenesis studies and NMR spectroscopic studies of the PSI - cytochrome interaction. This will also help to understand the different interaction in cyanobacteria and plants resulting from the insertion of a positive domain in PSI in an evolutionary event.

Comparison of the [2Fe-2S]-ferredoxin structure to that of a [4Fe-4S] ferredoxin from the hyperthermophilic bacterium Thermotoga maritima (Sticht et al., 1996) revealed that different structural features contribute to the thermostability in these proteins. Future mutational studies are intended to investigate the dynamics of both proteins by 15N relaxation measurements. While some ferredoxins have evolved towards stability at high temperatures, another group of ferredoxins shows the ability to maintain their structure at extremely high salt concentrations of up to 5 M. In this context, we are currently investigating the structural basis of the halophilic adaption of the [2Fe-2S] ferredoxin from Halobacterium salinarum, an extremely halophilic Archaeon. The 1H, 15N and 13C resonances of the protein were assigned (Schweimer et al., 2000) and allowed the calculation of a first structural model that is currently refined. A comparison of the structure to those of thermostable ferredoxins will help to answer the question about the existence of possible common structural principles of halophilism and thermostability.

The structure of cytochrome c6 gave us first insight into the molecular evolution of pro- and eukaryotic cytochromes (Beissinger et al., 1998). Additional information about the evolution of metalloproteins could be obtained from the structure of Guillardia theta rubredoxin. The structure of this protein (Schweimer et al., submitted), which is the first known structure of an eukaryotic rubredoxin, revealed a surface potential that differs considerably from that found in prokaryotic rubredoxins.

Literature:

• Sutter, M., Sticht, H., Schmid, R., Hörth, P., Rösch, P. & Haehnel, W. (1995)
  Cytochrome C6 from the thermophilic Synechococcus elongatus
  Photosynthesis: from Light to Biosphere, Vol II (Mathis, P., Ed.), Kluwer Academic Publishers, 563-566.
  
  
• Sticht, H., Wildegger, G., Baumann, B., Bentrop, D., Darimont, B. Sterner, R., Sutter, M., Haehnel, W. & Rösch, P. (1995)
  The Solution Structure of Iron Sulfur Proteins.
  Proceedings of the International Conference on Molecular Structural Biology (Kungl, A.J., Andrew, P.J. & Schreiber, H., Eds.), 73-82.
  
  
• Sticht, H., Wildegger, G., Bentrop, D., Darimont, B., Sterner, R. & Rösch, P. (1996)
  An NMR-derived model for the solution structure of oxidized Thermotoga maritima 1[4Fe-4S] Ferredoxin
  Eur. J. Biochem. 237, 726-735. [Abstract]
  
  
• Baumann, B., Sticht, H., Schärpf, M., Sutter, M., Haehnel, W. & Rösch, P. (1996)
  Structure of Synechococcus elongatus [2Fe-2S] Ferredoxin in Solution.
  Biochemistry 35, 12831-12841. [Abstract]
  
  
• Beißinger, M., Sticht, H., Sutter, M., Ejchart, A, Haehnel, W. & Rösch, P. (1998)
  The solution structure of Cytochrome C6 from the thermophilic Synechococcus elongatus
  EMBO J. 17, 27-36. [Abstract]
  
  
• Sticht, H. & Rösch, P. (1998)
  The structure of iron-sulfur proteins
  Prog. Biophys. Mol. Biol., 70, 95-136. [Abstract]
  
  
• Sticht, H. (1999)
  The structure of small electron transfer proteins
  Recent Research Developments in Biochemistry, in press
  
  
• Wastl, J., Sticht, H., Maier, U.-G., Rösch, P. & Hoffmann, S. (2000)
  Identification and characterization of a eukaryotically encoded rubredoxin in a crytomonad alga, submitted
  
  
• Schweimer, K., Hoffmann, S., Wastl., J., Maier, U.-G., Rösch, P. & Sticht, H. (2000)
  Solution structure of a zinc substituted rubredoxin from the cryptomonad Guillardia theta, submitted
  
  
• Schweimer, K., Marg, B.-L., Oesterhelt, D., Rösch, P. & Sticht, H. (2000)
  Sequence-specific 1H, 13C and 15N resonance assignments and secondary structure of [2Fe-2S] ferredoxin from Halobacterium salinarum, submitted