Radical S-adenosyl methionine (SAM) enzymes make up one of the largest and most functionally diverse superfamilies of enzymes that we currently know of.1 Enzymes in this family utilize a 4Fe-4S cluster and SAM to catalyze a wide variety of reactions that are critical across all domains of life. Some of such reactions include heme biosynthesis, vitamin biosynthesis, DNA repair, and anaerobic glucose metabolism. In 2021, radical S-adenosyl domain 1 (Rsad1), a radical SAM enzyme found in humans, was shown to have increased expression levels in Alzheimer’s diseased brain tissue samples when compared to cognitively normal brain tissue samples.2 The exact role of Rsad1 remains unknown, and biochemical investigation into Rsad1’s function stands to elucidate its biological function and potential relevance to Alzheimer’s disease. Rsad1 genes from two different mammalian sources have been cloned and inserted into prokaryotic expression vectors. These constructs allow overexpression in E. coli, and the resulting proteins have been purified to homogeneity. The Rsad1 protein purifies with variable amounts of iron bound, based on UV-visible spectroscopy and atomic absorption analysis. Reconstitution with iron and sulfide results in incorporation of a [4Fe-4S] cluster into the protein, as evidenced by iron and sulfur analyses and UV-vis spectroscopy. Treatment of the protein with dithionite results in reduction of the cluster to the [4Fe-4S]+ state which gives rise to a characteristic electron paramagnetic resonance (EPR) signal. Interestingly, Rsad1 has been shown to also bind protoporphyrin IX, potentially suggesting a role in heme transport and homeostasis, which is intricately linked to Alzheimer’s disease. Biochemical and spectroscopic characterization of this fascinating protein will be presented. This research provides the groundwork for further investigation into the connection between Rsad1 and Alzheimer’s disease and aids in furthering our knowledge of the biochemical progression of the disease.