Iron-Sulfur clusters (ISCs) are essential cofactors in biology that serve a variety of roles in many cellular processes. These roles include activating key enzymes involved in several critical pathways, including metabolism and ATP production. Assembly of ISCs occurs through the highly coordinated activities of core ISC biogenesis proteins ISD11, ISCU, Nsf1, and Frataxin (FXN). Defect or deficiency of these core proteins results in impaired energy metabolism and manifests in devastating human diseases such as: the fatal heart failure (HF) in patients with Friedreich's Ataxia caused by insufficient levels of he FXN; and the severe myopathy in patients deficient in ISCU. Therefore, a deeper understanding of the process of ISC biogenesis is necessary to understand the role this pathway plays in human disease. The molecular underpinnings of ISC biogenesis regulation, however, remain poorly understood. The long-term goal of this project is to better understand ISC biogenesis regulation, particularly in how ISC biogenesis is coordinated in with changes in metabolism and energy demands. Lysine acetylation and its regulation through the mitochondrial NAD+-dependent protein deacetylase Sirtuin 3 (SIRT3) is emerging as a major regulator of energy homeostasis. SIRT3's deacetylase activity modulates many metabolic enzymes involved in ATP production. Recent studies have demonstrated that SIRT3 deacetylase activity is necessary for maintaining ATP levels in cardiac tissue-underscoring the important role of SIRT3 in regulating mitochondrial energy metabolism. Proteomic studies identified several ISC biogenesis proteins as candidates of regulation by SIRT3, but the role of acetylation in regulating this critical celluar process is unknown. We predict that acetylation of proteins in the ISC biogenesis pathway serves as a mechanism of regulating mitochondrial energy homeostasis, as has been demonstrated for fatty- acid metabolic pathways and the TCA cycle. We identified one core ISC biogenesis protein that is a strong candidate in this manner. Our central hypothesis is that acetylation and deacetylation by SIRT3 is critical in modulating enzymatic function, thereby regulating ISC biogenesis in a novel manner. We will test our central hypothesis by pursuing the following two specific aims: (1) determine the biochemical role of acetylation on the enzymes involved in ISC biogenesis, and; (2) determine the role of SIRT3 in regulating ISC biogenesis in vivo. Our findings will provide novel insights into an unexplored area of science: a crosstalk between acetylation and ISC biogenesis, and its role in energy homeostasis. Additionally, our findings could identify a new strategy for the development of therapies for diseases of impaired energy production, such as HF, and ISC-related disorders.