Biochemical and Biophysical Characterization of Ferrous Iron Transport Proteins FeoA and FeoB


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Chemistry & Biochemistry



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Iron is indispensable to nearly all forms of life and is involved in diverse and essential chemical processes, such as aerobic cellular respiration, N2 fixation, gene regulation, and DNA biosynthesis. For infectious organisms, iron acquisition systems are commonly associated with the intracellular growth, survival, and virulence of pathogens, which are able to modify their iron uptake strategies dynamically in response to changing host environments. The past few decades have been marked with an increased understanding of pathogenic ferric iron (Fe3+) and heme acquisition. In contrast, though the necessity of ferrous iron (Fe2+) transport for pathogenesis has been established, the precise details of this process remain enigmatic, and Fe2+ transporters remain unexploited as drug targets to combat drug-resistant organisms. The ferrous iron transport (Feo) system, canonically comprising the proteins FeoA, FeoB, and FeoC, has been identified as the predominant prokaryotic Fe2+ transport system. FeoA and FeoC are cytoplasmic proteins of unknown functions, and FeoB is the transmembrane component responsible for Fe2+ import. Interestingly, multiple variations on this operonic arrangement are found in pathogens, which also remain uncharacterized, including a naturally-occurring FeoAB fusion protein. In this work, we undertake several biochemical and biophysical approaches to determine the function of FeoA and its potential protein-protein interactions with the soluble domain of FeoB. We show that FeoA serves to regulate the GTPase activity of FeoB and propose a model for Fe2+ import. Additionally, we present an improved expression and purification system for intact FeoB, that can be applied to FeoB and FeoAB of differing protein topologies. Lastly, we sought the characterize the metal-binding and transport capabilities of FeoB by designing an in vitro liposome-based assay. Combined, our results increase our understanding of Feo function and lay the groundwork for future studies of this critical prokaryotic Fe2+ transport system.