Characterisation of haem lyases and their dedicated roles in bacterial cytochrome c biogenesis

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Background: C-type cytochromes are a widespread class of proteins that play a vital role in the energy-conserving metabolism of prokaryotic and eukaryotic organisms including many pathogens. The key feature in cytochrome c biogenesis is the covalent attachment of the haem cofactor to two (or rarely only one) cysteine residues arranged in a haem c binding motif, typically CXXCH. This reaction is catalysed by the membranebound enzyme haem lyase. No bacterial haem lyase has ever been purified and its reaction mechanism is unknown. Two independent enzymic systems catalysing bacterial cytochrome c biogenesis have been described but only the Ccm system (or system I) of Escherichia coli has been investigated in any detail. Goals: This project aims to reveal the function of haem lyases involved in the CcsA system (or system II). The arguably best-suited model bacterium is Wolinella succinogenes. Its genome encodes three distinct haem lyases that are expected to differ in their specificity towards distinct haem c binding motifs. It will be examined whether or not W. succinogenes wild-type and haem lyase gene deletion mutants are able to covalently attach haem to a model c-type cytochrome containing either the classical or various unconventional haem c binding motifs. One haem lyase (NrfI) has already been shown to recognise a unique CXXCK haem c binding motif of pentahaem cytochrome c nitrite reductase. NrfI will be subject to site-directed modification in order to identify essential amino acid residues. Overproduction, purification and biochemical characterisation of system II haem lyases will be attempted. Impact: The proposed work will broaden the general knowledge on posttranslational protein maturation and prediction of haem c binding motifs. Furthermore, it aims to establish W. succinogenes as host for efficient c-type cytochrome production. Key words: Cytochrome c maturation; Cytochrome c haem lyase (CCHL); Haem c binding motif; Wolinella succinogenes; Bacterial energy metabolism.