Abstract 1359 Feo-mediated prokaryotic ferrous iron transport: the delicate dance of FeoA and FeoB

The acquisition of iron is essential to establishing virulence among most pathogens. Under acidic and/or anoxic conditions, most bacteria utilize the widely distributed ferrous iron (Fe2+) uptake (Feo) system to import metabolically required iron. The Feo system is inadequately understood at the atomic, molecular, and mechanistic levels, but we do know it commonly comprises a main membrane component (FeoB) that has NTPase activity and is essential for iron translocation, and a small, cytosolic protein (FeoA) with an essential but unknown function. In this work, we characterize a naturally occurring FeoA-NFeoB fusion to decipher FeoA function. We have used small-angle X-ray scattering (SAXS) to show that NFeoB and FeoA do not interact in the nucleotide-free form. In contrast, SAXS data of nucleotide-bound FeoA-NFeoB suggest a nucleotide-dependent interaction. We have mapped this protein-protein interface using hydrogen-deuterium exchange (HDX) mass spectrometry in addition to solving the X-ray crystal structure of the FeoA domain of this fusion to 1.6 Å resolution. Intriguingly, enzymatic characterization of this fusion protein displays the slowest GTP hydrolysis to-date of an NFeoB construct. Combined, these data suggest the role of FeoA is to prevent GTP hydrolysis, which could lock FeoB in an open or "on" state to allow for the translocation of ferrous iron into the prokaryotic cytosol. This work was supported by the NIH grant R35 GM133497