Iron-sulfur (Fe-S) clusters are protein cofactors that are found in all living organisms and have a variety of functions including electron transfer, substrate activation, and regulation of gene expression. In humans, the synthesis of Fe-S clusters primarily occurs in the mitochondria and requires an assembly complex composed of NFS1, ISD11, ISCU2, and FXN subunits. NFS1 and ISD11 function as a cysteine desulfurase to deliver sulfur to ISCU2 where iron and electrons (from ferredoxin) are used to build a cluster. This process appears to be regulated by FXN. Importantly, mutations in any of these genes are related to human disease. Currently, limited information is available about the assembly complex; therefore, to determine structural information about the assembly of the complex, we implemented a method to investigate protein-protein interactions using hydroxyl radical footprinting of solvent-exposed amino acid residues. We designed a flow cell and optimized the conditions for amino acid oxidation by hydroxyl radicals, which originate from the homolytic cleavage of hydrogen peroxide by a 248 nm KrF laser. The proteolytic fragmentation of the Fe-S assembly proteins was optimized to maximize the amino acid coverage observed by mass spectrometry. MALDI and LC-ESI mass spectrometry methods were explored to determine the ideal way to detect oxidation of the proteins. Alternate methods to explore structure and function were also employed and included mutagenesis, activity assays and spectroscopic methods. Together, these results contribute to our understanding of the eukaryotic Fe-S assembly complex with potential implications in human disease.

Hydroxyl radical footprinting of the human Fe-S cluster assembly complex

251st National Meeting (2016)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.