Heterologous Production and Characterization of Multiheme Cytochrome c Enzymes

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Multiheme Cytochromes c are a diverse family of electron carriers and redox enzymes that play a central role in several metabolic pathways of the biogeochemical nitrogen cycle. Their heme cofactors pack into conserved interaction motifs that allow for strong electronic cou-pling and efficient electron transfer, but thus also complicate spectroscopic investigations. The enzymes nitrite reductase, hydroxylamine oxidoreductase and hydrazine oxidoreductase share a highly conserved heme group arrangement and a similar architecture of the respec-tive active site, yet they catalyze very different chemistry. Knowledge about these systems is on very different levels, but to date no concise, comparative characterization of their sub-strate and product ranges has been carried out. We thus propose to combine the cyto-chromes c already under study in our laboratories with novel enzymes for which production protocols will be established and to elevate our knowledge on these systems to a common level that includes (i) the establishment of gene expression protocols for recombinant cyto-chrome c production, (ii) functional assays to probe the substrate and product range, (iii) structural characterization by X-ray crystallography and (iv) functional modification by site-directed mutagenesis. Single crystal UV/vis and EPR spectroscopy will be used to identify individual heme groups and to study intramolecular electron transfer events.