Heterologous Production and Characterization of Multiheme Cytochrome c Enzymes

• Applicants: Professor Dr. Oliver Einsle; Professor Dr. Jörg Simon
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2010 to 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 186439814

• Final Report Year: 2019

Final Report Abstract

Multiheme Cytochromes c are a diverse family of electron carriers and redox enzymes that play a central role in several metabolic pathways within the biogeochemical nitrogen cycles of nitrogen, sulfur and iron. Their heme cofactors pack into conserved interaction motifs that allow for strong electronic coupling and efficient electron transfer. In this project, various multiheme cytochromes c have been recombinantly produced in either Escherichia coli or Wolinella succinogenes cells using sophisticated gene expression and cytochrome c biogenesis systems. The studied cytochromes c included diheme cytochrome c peroxidases, pentaheme cytochrome c oxidoreductases of the NrfA family, octaheme cytochrome c oxidoreductases of the Hao and MccA families and the nonaheme cytochrome c OcwA involved in metal reduction by Gram-positives. The cytochromes were purified and characterized in terms of enzyme activities, substrate/product range, spectroscopic features, high-resolution crystal structures and evolutionary implications. The results contributed to our understanding of the structure/function relationships of periplasmic/extracellular cytochrome c-dependent electron transport networks that are crucial for many environmentally significant processes.

Publications

• (2011) Physiological function and catalytic versatility of bacterial multihaem cytochromes c involved in nitrogen and sulfur cycling. Biochem. Soc. Trans. 39, 1864-1870
  Simon, J., Kern, M., Hermann, B., Einsle, O. & Butt, J.N.
  
• (2011) The oxidative and nitrosative stress defence network of Wolinella succinogenes: cytochrome c nitrite reductase mediates the stress response to nitrite, nitric oxide, hydroxylamine and hydrogen peroxide. Environ. Microbiol. 13, 2478-2494
  Kern, M., Volz, J. & Simon, J.
  
• (2012) Enzyme or Electrode? Structure 20, 1132-1134
  Einsle, O.
  
• (2012) MacA is a second cytochrome c Peroxidase in Geobacter sulfurreducens. Biochemistry 51, 2747-2456
  Seidel, J., Hoffmann, M., Ellis, K.E., Seidel, A., Spatzal, T., Gerhardt, S., Elliott, S.J. & Einsle, O.
  
• (2013) Structure of the processive rubber oxygenase RoxA from Xanthomonas sp. Proc. Natl. Acad. Sci. USA 110, 13833-13838
  Seidel, J., Schmitt, G., Hoffmann, M., Jendrossek, D. & Einsle, O.
  
• (2014) The production of ammonia by multiheme cytochromes c. In: The Metal-Driven Biogeochemistry of Gaseous Compounds in the Environment, Eds P.M.H. Kroneck, M.E. Sosa Torres. Met. Ions Life Sci. 14, 211-236
  Simon, J. & Kroneck, P.M.H.
  
• (2015) Multiheme Peroxidases. In: Heme Peroxidases (Raven, E.L. & Dunford, H.B., eds.) RSC Publishing, Oxford, 113-132
  Brausemann, A., Seidel, J., Wüst, A. & Einsle, O.
  
• (2015) Resolution of key roles for the distal pocket histidine in cytochrome c nitrite reductases. J. Am. Chem. Soc. 137, 3059-3068
  Lockwood, C.W.J., Burlat, B., Cheesman, M.R., Kern, M., Simon, J., Clarke, T.A., Richardson, D.J. & Butt, J.N.
  
• (2015) The octahaem MccA is a haem c–copper sulfite reductase. Nature 520, 706-709
  Hermann, B., Kern, M., La Pietra, L., Simon, J. & Einsle, O.
  
• (2017) Epsilonproteobacterial hydroxylamine oxidoreductase (εHao): Characterization of a 'missing link' in the multihaem cytochrome c family. Mol. Microbiol. 105, 127-138
  Haase, D., Hermann, B., Einsle, O. & Simon, J.
  
• (2017) NMR studies of the interaction between inner membrane-associated and periplasmic cytochromes from Geobacter sulfurreducens. FEBS Lett. 591, 1657-1666
  Dantas, J.M., Brausemann, A., Einsle, O. & Salgueiro, C.A.
  
• (2017) Structural and functional analysis of latex clearing protein (Lcp) provides insight into the enzymatic cleavage of rubber. Sci. Rep. 7, 6179
  Ilcu, L., Röther, W., Birke, J., Brausemann, A., Einsle, O. & Jendrossek, D.
  
• (2019) How Thermophilic Gram-Positive Organisms Perform Extracellular Electron Transfer: Characterization of the Cell Surface Terminal Reductase OcwA. mBio 10, e01210-e01219
  Costa, N.L., Hermann, B., Fourmond, V., Faustino, M.M., Teixeira, M., Einsle, O., Paquete, C.M. & Louro, R.O.