Iron-sulfur clusters, an important class of redox active cofactors, are ligated by protein-based Cys ligands in a variety of nuclearities. Traditionally, these clusters serve as one-electron transfer units, though many clusters are capable of catalytic activity and sensing functions. Recently, a greater number of iron-sulfur clusters with non-Cys ligation have been identified, wherein one or more of the Cys ligands are replaced by an alternative amino acid residue such as His or Asp. In most cases the role of this ligand substitution is unknown. Some hypotheses are that non-Cys ligation may modify reduction potential, allow for proton-coupled electron transfer, or modulate cluster stability. The human mitoNEET protein contains a 1-His, 3-Cys ligated [2Fe-2S] cluster, identified by the presence of a CDGSH peptide motif. MitoNEET is a binding target for the type II-diabetes drug, pioglitazone, and is implicated in controlling mitochondrial iron levels. How exactly mitoNEET functions in the cell is unknown, as is the role its uniquely ligated FeS cluster may play. This thesis uses mitoNEET as a model for the study of non-Cys ligated FeS clusters and their biological function. Protein film voltammetry was used to examine the pH-dependent electrochemical properties of the mitoNEET cluster, indicating that multiple as yet unidentified protonations control redox potential and that drug binding impacts cluster reduction and protonation. Additionally, the effect of reduction and protonation on cluster and protein structure instability was examined through absorbance and circular dichroism measurements, suggesting an important role for cluster lability in protein function. The CDGSH-motif family of [2Fe-2S] cluster-binding proteins was examined using protein similarity networks. This technique highlights the evolutionary relationship among these proteins, and has led to further work examining the DUF1271 domain containing proteins E. coli YjdI and A. vinosum Alvin0680 (a CDGSH-DUF1271 fusion). This work furthers the scientific knowledge of non-Cys ligated Fe-S clusters by improving our understanding of how the mitoNEET His-ligand contributes to proton-coupled electron transfer and cluster instability, and how the broader class of CDGSH-motif proteins is organized.

NEET proteins belong to a unique class of [2Fe-2S] cluster proteins that have been shown to participate in various biological processes such as regulating iron, reactive oxygen species and apoptosis within the cell and are localized to the mitochondria. Disruption of the mitochondrial NEET proteins are associated with different human diseases such as obesity, neurodegeneration, cancer and diabetes. In humans, a missense mutation in the CISD2 gene results in a heritable multisystem disorder termed Wolfram syndrome 2 (WFS2), a disease which displays an early onset of juvenile diabetes and various neuropsychiatric disorders. The C. elegans genome contains three previously uncharacterized cisd genes: cisd-1, which has homology to the human CISD1 and CISD2, and cisd-3.1 and cisd-3.2, both of which have homology to the human CISD3. Disruption of the cisd-3 gene(s) function results in mis-regulation of proteostasis in the mitochondria, whereas cisd-1 and cisd-3.1 disruption impacts proteostasis in the endoplasmic reticulum. Reduction of cisd-3.2 gene function also leads to a developmental delay in C. elegans. A knockout mutation of the cisd-3.2(pn68) gene function results in various germline defects including delayed development progression and morphological defects. Furthermore, I show the cisd gene(s) and protein expression profiles is present relative to sex, tissue type and developmental stages. This work is significant because it provides further insight of the essential role of CISD-3 relative to C. elegans. Furthermore, my studies can contribute to new genetic discoveries that will widen the scientific research relative to NEET protein family studies.