Project Summary/Abstract: Metalloenzymes perform chemical transformations with rates and selectivities that remain the envy of synthetic chemists. By definition these transformations utilize earth-abundant transition metals and environmentally friendly reagents. Furthermore, while some metalloenzymes utilize specialized cofactors, many are able to achieve these transformations using the relatively limited natural ligand set provided by the amino acids. Indeed, in many cases a single coordination motif is used to promote a variety of mechanistically distinct transformations providing evidence for the important of the secondary and tertiary structure of the protein environment for dictating reaction mechanism. One approach to understanding the structure-function principles is to de novo design metalloenzymes from scratch. Herein we exploit de novo protein design to allow us to systematically alter the local environment around a biologically important, ambiphilic reaction intermediate, the ferric superoxo. We then seek to utilize this understanding and the newfound ability to design specific small molecule binding proteins to explore physiologically important C– H activation reactions at a mononuclear, non-heme Fe center. C–H activation reactions are of particular interest from a structure-function perspective because their success has been shown to be highly dependent on substrate positioning, thereby providing a sensitive test of our ability to de novo design binding pockets. Improving our ability to design controlled binding pockets would open the possibility for many applications of de novo proteins. The proposed studies will primarily be achieved using optical spectroscopy to characterize the electronic structure and thermochemistry of the different species. These studies will be complemented by structural information derived from multinuclear NMR spectroscopy and X-ray crystallography. The results would represent a breakthrough in protein design with implications for fundamental understanding of how metalloproteins utilize simple ligand sets to generate and harness reactive intermediates for achieving chemically challenging transformations.