PROJECT SUMMARY/ABSTRACT Metalloproteins carry out many cellular functions that are central to biology and human health, While our knowledge of how metalloproteins function has grown immensely thanks to technological advances, we still possess only a superficial understanding of the interplay between protein structure/dynamics and metal coordination/reactivity, As a result, it has been challenging or even impossible (a) to predict the functional mechanism of metalloproteins simply by looking at their structures, (b) to emulate or improve upon the structures and functions of metalloproteins by de nova design, and (c) to understand how complex bioinorganic functions may have emerged on simple peptide/protein scaffolds during natural evolution, The overarching goal of the proposed research program is to address these three challenges by designing and constructing protein scaffolds with complex metal-based functions from scratch, Toward this end, we have recently developed/adapted two powerful approaches to metalloprotein design, Metal-Centered Protein Assembly (MCPA) and MachineLearning- guided Design of Metalloproteins (MLDM), which allow novel protein structures to be built around metal active sites rapidly and with atomic accuracy, In the proposed research, we will further develop these "metalcentered" protein design strategies (and establish their generalizability) by constructing de nova protein scaffolds that will provide access to diverse metal active sites with tunable primary coordination spheres, secondarysphere environments and global structures/properties that are difficult to attain with other protein design strategies, We will use these protein scaffolds to build new metalloenzymes for challenging ester, amide and phospho-ester bond hydrolysis reactions (Specific Aim 1 ), for redox reactions involving dioxygen binding and activation (Specific Aim 2), and for abiological catalytic transformations (e,g,, hydride and carbene transfer) (Specific Aim 3), These efforts will uncover fundamental structure-function relationships that govern diverse metalloprotein activities, lead to better understanding of how bioinorganic complexity arises in simple protein scaffolds, and furnish new-to-nature reactivities.