Project Summary/Abstract Metalloproteins perform chemical transformations with rates and selectivites that have yet to be achieved in synthetic or designed systems. These differences in reactivity are directly linked to the environment produced by the protein matrix. To test our understanding of how metalloproteins function, I aim to design de novo metalloenzymes from scratch. Proteins that bind porphyrin-like cofactors are of particular interest, as heme proteins are known to perform a variety of reactions. Recently, I designed a protein to bind the abiological porphyrin, Mn-diphenylporphyrin (MnDPP), that provided the first crystallographic structure of a de novo designed porphyrin-binding protein (MPP1). MPP1 was also capable of stabilizing a Mn(V)-oxo species, a powerful oxidant that can perform sulfoxidation of thioether substrates. The proposed research seeks to elucidate design features necessary to control the reactivity/stability of this high-valent species through rational mutagenesis of my designed protein. This will allow direct correlation of changes in reactivity to changes in structure. To gain greater control of substrate orientation and, therefore, product distribution, I will design a 5-helix bundle that has a large pocket for substrate binding. Using the design strategy for MPP1 and in-house developed computational methods, the 5-helix bundle will be parameterized from scratch and designed to bind MnDPP. A library of sequences will be expressed and screened using high-throughput methods for binding and sulfoxidation activity. Promising scaffolds will then be redesigned to include substrate-specific interactions to bind the anti-inflammatory drug, diclofenac. Using COMBS, a recently developed bioinformatics method for designing backbone specific polar interactions, I will design two proteins to control the orientation of diclofenac to direct the hydroxylation to yield 5-hydroxydiclofenac or 4’- hydroxydiclofenac. This work would be a breakthrough in protein design and will directly impact the fundamental understanding of the effects of protein environments on the function of metal centers in metalloproteins.