Assaying the metal-specificity of heme biosynthesis enzyme variants | Poster Board #744

Cofactors are organic and inorganic molecules that attach to a protein to enable its specific function. Heme is an inorganic cofactor composed of a protoporphyrin IX (PPIX) ring with a central iron atom. Other metals, such as cobalt, nickel, and copper, have been artificially inserted in place of iron within heme proteins, changing the properties of the protein and enabling new functions. These metal-substituted heme proteins have been synthesized semi-biosynthetically, but a purely biosynthetic route to their production would make the exploration of their abilities more efficient and broaden their potential applications. The overall aim of this study is to efficiently assay variants of ferrochelatase, the last enzyme in the heme biosynthesis pathway that catalytically inserts iron into PPIX, to discover variants that can generate metal-substituted hemes in E. coli. The expression of K. pneumonia ferrochelatase has been tested in E. coli, while protocols for its currently under development. An efficient, quantitative assay for the metal-insertion reaction catalyzed by ferrochelatase has been developed that involves spectral decomposition of spectral data generated by a plate-reader. In addition, all plasmids have been successfully cloned and their sequences confirmed via Sanger Sequencing for the next phase of the project, which will assay the catalytic attachment of these biosynthetic metal-substituted hemes to horse cytochrome c using variants of holocytochrome c synthase.