Characterisation of haem lyases and their dedicated roles in bacterial cytochrome c biogenesis
Final Report Abstract
This project dealt with the cytochrome c biogenesis system II of the Epsilonproteobacterium Wolinella succinogenes and focussed on the substrate specificity of three distinct cytochrome c haem lyase (CCHL) enzymes and the implementation of a genetic system enabling heterologous overproduction of multihaem cytochromes c in W. succinogenes. In the following, the most important results of the project are briefly described. (I) The function of the three CCHL isoenzymes (NrfI, CcsA1 and CcsA2) was clarified. NrfI recognizes the unconventional haem c binding motif CX2CK found in the active site of cytochrome c nitrite reductase (NrfA) whereas CcsA1 is responsible for haem c attachment to the novel octahaem cytochrome c MccA that contains an unprecedented CX15CH haem c binding motif. CcsA2 attaches haem to the standard CX2CH haem c binding motif. This was shown experimentally by producing active CcsA2 in Escherichia coli. (II) Extensive site-directed modification of all three CCHLs was performed in W. succinogenes and in E. coli and the CCHL variants were tested towards their activity in cytochrome c maturation. Essential histidine residues as well as a tryptophan-rich domain were shown to be essential. These data support the now well-established model that the large membrane-bound system II CCHLs from Epsilonproteobacteria are involved in both haem b export and haem c attachment to apo-cytochrome c. (III) In collaboration with the lab of Robert Kranz (St. Louis, USA), all three CCHLs from W. succinogenes were produced in E. coli but only CcsA2 could mature a reporter cytochrome c. These findings implied that NrfI and CcsA1 recognize as yet unknown features within their cognate apo-cytochromes in addition to the respective unconventional haem c binding site. (IV) A convenient and easy-to-use system for heterologous cytochrome c production in W. succinogenes was established and successfully tested. It is the first such system that is based on the bacterial cytochrome c biogenesis system II. The NrfA enzymes from Campylobacter jejuni and E. coli were produced in large amounts and found to be highly active. This approach is considered a valuable alternative to cytochrome c overproduction in E. coli. Overall, the outcome of this project deepened our understanding of cytochrome c maturation which is an essential process in bacterial energy metabolism and indispensable for many substrate conversions within Earth’s biogeochemical nitrogen, sulfur and iron cycles.
Publications
- (2006) Multiple haem lyase genes indicate substrate specificity in cytochrome c biogenesis Biochem. Soc. Trans. 34, 146-149
R.S. Hartshorne, D.J. Richardson, J. Simon
- (2007) A dedicated haem lyase is required for the maturation of a novel bacterial cytochrome c with unconventional covalent haem binding Mol. Microbiol. 64, 1049-1060
R.S. Hartshorne, M. Kern, B. Meyer, T.A. Clarke, M. Karas, D.J. Richardson, J. Simon
- (2009) Electron transport chains and bioenergetics of respiratory nitrogen metabolism in Wolinella succinogenes and other Epsilonproteobacteria. Biochim. Biophys. Acta 1787, 646-656
M. Kern, J. Simon
- Substrate specificity of three cytochrome c haem lyase isoenzymes from Wolinella succinogenes: unconventional haem c binding motifs are not sufficient for haem c attachment by NrfI and CcsA1. Mol. Microbiol 75, 122-137
M. Kern, F. Eisel, J. Scheithauer, R.G. Kranz, J. Simon
