STRUCTURAL & BIOPHYSICAL CHARACTERIZATIONS OF MEMBRANE-BOUND AND MEMBRANE-ASSOCIATED COMPONENTS OF THE FERROUS IRON TRANSPORT (FEO) SYSTEM

Abstract

Iron (Fe) is a vital transition metal for virtually all living organisms. In its ionic form, this element functions as a powerful cofactor within a multitude of enzymes responsible for a wide range of complex chemical reactions including, but not limited to, de novo DNA biosynthesis, central carbon metabolism, vitamin biogenesis, and even N2 fixation. Due to its critical necessity in these essential metabolic processes, organisms such as bacteria have a high demand for the uptake of iron. However, the mode of iron acquisition is linked to the oxidation state of the iron ion, which is a product of the prevailing environmental conditions. In acidic and reducing conditions commonly encountered by pathogenic bacteria within the human gut or locations within biofilms, ferrous iron (Fe2+) dominates the environment, but the precise mechanism by prokaryotes acquire Fe2+ remains unclear. In this dissertation, the structural and biophysical characteristics of the membrane-bound and membrane-associated components of the ferrous iron transport (Feo) system, the most widely distributed and the primary Fe2+ transport system utilized by bacteria, are investigated. First, structures of the N-terminal domain of FeoB (NFeoB) from Vibrio cholerae (VcNFeoB), the causative agent of the disease cholera, are presented for the first time and give critical insight into the surprising nucleotide promiscuity of FeoB. Next, in an attempt to provide homogeneous, near-native samples of full-length, intact FeoB for future structural characterization, V. cholerae FeoB (VcFeoB) was extracted directly from bacterial membranes in near native-like lipid environments through the use of styrene-maleic acid (SMA)-copolymers. SMA-extracted VcFeoB showed high purity, good monodispersity, and displayed differences in NTP-dependent activity in the presence of lipids compared to detergent-solubilized VcFeoB. Finally, a newly discovered single-pass transmembrane protein of the Feo system termed FeoD (formally FeoI) from Streptococcus thermophilus (StFeoD) was first modeled using AlphaFold3 before being cloned, expressed, purified, and partially characterized. Contrary to previous reports, we show that the Cys-rich StFeoD co-purifies with a partially bound [Fe-S] cluster, similarly to FeoC, and optimization and further characterization is currently underway. When taken all together, this dissertation deepens our understanding of the membrane-bound and membrane-associated components of the Feo system and paves the way for future structural work to understand the precise mechanism of prokaryotic Fe2+ transport.