The structure of a novel ferredoxin: FhuF, a ferric-siderophore reductase from E. coli K-12 with a novel 2Fe-2S cluster coordination

Iron is a vital element for life. However, after the Great Oxidation Event, the bioavailability of this element became limited. To overcome iron shortage and to scavenge this essential nutrient, microorganisms use siderophores, secondary metabolites that have some of the highest affinities for ferric iron. The crucial step of iron release from these compounds to be subsequently integrated into cellular components is mediated by Siderophore-Interacting Proteins (SIPs) or Ferric-siderophore reductases (FSRs). In this work, we report the structure of an FSR for the first time. FhuF from laboratory strain Escherichia coli K-12 is the archetypical FSR, known for its atypical 2Fe-2S cluster with the binding motif C-C-X10-C-X2-C. The 1.9 [A] resolution crystallographic structure of FhuF shows it to be the only 2Fe-2S protein known to date with two consecutive cysteines binding different Fe atoms. This novel coordination provides a rationale for the unusual spectroscopic properties of FhuF. Furthermore, FhuF shows an impressive ability to reduce hydroxamate-type siderophores at very high rates when compared to flavin-based SIPs, but like SIPs it appears to use the redox-Bohr effect to achieve catalytic efficiency. Overall, this work closes the knowledge gap regarding the structural properties of ferric-siderophore reductases and simultaneously opens the door for further understanding of the diverse mechanistic abilities of these proteins in the siderophore recycling pathway.