Natural hydrogenases catalyze the reduction of protons to molecular hydrogen reversibly under mild conditions. The relevance of this reaction to energy production, and in particular to sustainable fuel production, has spurred considerable interest around these enzymes. The catalytic site of hydrogenases, termed the H-cluster, is composed by a [4Fe4S] cluster and a [FeFe] site coordinated by a non-protein dithiolate bridging ligand as well as carbon monoxide (CO) and cyanide (CN) ligands. The site is anchored to the protein through a cysteine that bridges the proximal iron-sulfur cluster and one iron. Such an unusual cluster is assembled in vivo through a complex biosynthetic machinery. For these reasons, direct mimicking of natural hydrogenases has proven to be a daunting task.In our lab, we have taken a 'retrosynthetic' approach towards the construction of a functional mimic of [Fe-Fe] hydrogenases using de novo protein designing principle. To mimic the electron transfer domain, we designed  two [4Fe-4S] cluster binding sites into an existing protein, DSD, which is a de novo designed domain swapped dimer. The resulting protein, DSD-bis[4Fe-4S], contains two clusters as a distance of 36 �(1). On this scaffold, the distance between two clusters can be varied as desired using vertical translation along the axis of the coiled coil. To mimic the catalytic diiron site, we utilize an artificial amino acid containing a 1,3 dithiol moiety as an anchor to covalently secure the diiron�cluster at any position in an artificial protein scaffold (2). We verified the correct incorporation of the cluster into a model helical peptide UV-Vis, FTIR, ESI-MS and CD spectroscopy. Current design of more complex system is focused on de novo designed four-helix bundles that host the di-iron cluster inside the hydrophobic core. The full length four helix bundles are generated by bioorthogonal chemoselective ligation of an expressed three-helix segment containing p-acetylphenylalanine (p-AcF) to a synthetized single helix containing the 1,3-dithiol moiety, and functionalized with an hydroxylamine moiety. With these artificial proteins, it will be possible to explore the effect of second sphere interactions on the activity of the diiron center, and to include in the design properties such as compatibility with conductive materials and electrodes. References:1. Roy A., Serrau I., Astashkin A., and Ghirlanda, G.: �De novo design of an artificial bis-[Fe4S4]-binding protein� (2013) Biochemistry, 52, 7586-942. Roy A., Madden C. and Ghirlanda G*. �Photo-induced hydrogen production in a helical peptide incorporating a [FeFe] hydrogenase active site mimic. Chem Commun. (2012) 48(79):9816-8

De novo design and synthesis of peptide based functional mimics of [FeFe] hydrogenase

248th National Meeting (2014)

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