Project Summary This research program focuses on two inadequately understood metalloprotein systems that are linked to health and human disease and aims to uncover the mechanistic underpinnings of these essential biological processes. The first biological process of focus is bacterial ferrous iron (Fe2+) generation, acquisition, and sensing. The chief prokaryotic Fe2+ acquisition system is the Feo system, which is present in nearly all bacteria and is used by pathogens to establish infection in mammalian hosts. Our previous work has begun to unravel the mechanistic details of this important iron acquisition pathway. Importantly, there is an emerging connection between Feo and additional membrane-bound proteins that function more broadly in bacterial Fe2+ homeostasis through the sensing Fe2+ to control biofilm formation (BqsR/S) and the utilization of Fe3+-siderophores to supply Fe2+ to the Feo system (membrane ferric reductases or mFRs). This proposal outlines a comprehensive approach to study these three systems (Feo, Bqs, mFRs) both in vitro and in vivo. Leveraging structural, spectroscopic, and biochemical analyses, this proposal aims to define the mechanism of bacterial Fe2+ generation, acquisition, and sensing, which will position future researchers to explore the urgent but broadly impactful possibility that these systems may be exploited to combat bacterial virulence. The second biological process of focus is eukaryotic post-translational arginylation, catalyzed by the enzyme arginyltransferase 1 (ATE1). ATE1-catalyzed arginylation typically targets the N-terminus of proteins, altering protein function and fate in vivo. Normal ATE1 activity is critical for neurogenesis, cardiovascular development, cancer, and viral infections, but the structural and mechanistic details of ATE1-mediated arginylation are sorely lacking, prohibiting the targeting of this system for therapeutic intervention. Our previous work has uncovered the structure of yeast ATE1 for the first time, has shown that ATE1s are O2-sensitive [Fe-S] proteins, and has developed a mechanistic framework for post-translational arginylation. This proposal aims to uncover the structure of the arginylation complex and its link to O2 sensitivity, to determine the structure of a mammalian ATE1, and to understand the evolution of ATE1. To achieve this goal, this proposal combines in vitro and in vivo structural, biochemical, and functional methods to elucidate the components of post-translation arginylation in order to design small molecules that target ATE1 for intervention. Combined, the results from this proposal hold the promise to aid in the development of therapeutics to abrogate bacterial virulence linked to iron homeostasis and to treat cellular diseases linked to post-translational arginylation.