UC San Diego

Iron-sulfur cluster proteins are considered to be one of the most ubiquitous prosthetic groups since ancient times. Regulations of iron homeostasis through iron-sulfur proteins is of critical importance for cell development as free labile iron can cause DNA and lipid damage, lesions and ultimately cancer from production of reactive oxygen species (ROS). The unique NEET class of proteins harbors 3 Cys- 1 His ligand configuration having a consensus sequence of [C-X-C-X2-(S/T)-X3-P-X-C-D-G-(S/A/T)-H] that houses 2Fe-2S clusters. This thesis focuses on a monomeric human NEET protein MiNT found within the mitochondrial matrix as opposed to the dimeric mNT and NAF-1. Though MiNT has an upregulated expression in cancer cells and 2Fe-2S cluster properties are similar to mNT and NAF-1, not much is known about it. Characterization of MiNT’s structure function relationships are studied in subsequent chapters in order to understand the effects of its asymmetry by doing a point mutation on the histidine at position 75 and 113 individually and both. Ultra-violet visible spectroscopy (UV-Vis) and circular dichroism (CD) will be employed to further understand MiNT’s unique differences as a monomeric protein carrying both clusters. These assays show that there are effects on the stability by temperature and structure differences are analyzed through the differences of the CD spectra. Comparison of spectra between oxidized and reduced state in UV-Vis for all constructs show that a possible tryptophan residue is responsible for acting as an electron transfer hub between the two clusters in MiNT, not noticed in the other human NEET proteins.