A unique ferrous iron binding mode is associated with large conformational changes for the transport protein FpvC of Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa secretes pyoverdine, a major siderophore to get access to iron, an essential nutrient. Pyoverdine scavenges ferric iron in the bacterial environment with the resulting complex internalized by bacteria. Releasing of iron from pyoverdine in the periplasm involves an iron reduction by an inner membrane reductase and two solute‐binding proteins (SBPs) FpvC and FpvF in association with their ABC transporter. FpvC and FpvF belong to two different subgroups of SBPs within the structural cluster A: FpvC and FpvF were proposed to be a metal‐binding protein and a ferrisiderophore‐binding protein respectively. Here, we report the redox state and the binding mode of iron to FpvC. We first solved the crystal structure of FpvC bound to a fortuitous Ni2+ by single anomalous dispersion method. Using a different protein purification strategy, we determined the structure of FpvC with manganese and iron, which binds to FpvC in a ferrous state as demonstrated by electron paramagnetic resonance. FpvC is the first example of a hexahistidine metal site among SBPs in which the Fe2+ redox state is stabilized under aerobic conditions. Using biophysics methods, we showed that FpvC reversibly bind to a broad range of divalent ions. The structure of a mutant mimicking the apo FpvC reveals a protein in an open state with large conformational changes when compared with the metal‐bound FpvC. These results highlight that the canonical metal site in FpvC is distinct from those yet described in SBPs and they provide new insights into the mechanism of PVD‐Fe dissociation in P. aeruginosa.

Structural and thermodynamic insights into a novel Mg2+–citrate-binding protein from the ABC transporter superfamily

“…Notably, the thermodynamic data indicate a comparatively lower affinity of MctA towards Mg 2+ compared with other divalent metal ions. A comparable lower affinity of the physiological metal ions has also been reported previously for Mn 2+ and Fe 2+ (Coun ˜ago et al, 2014;Vigouroux et al, 2020). This is probably to facilitate the release of the substrate to the transmembrane domains (TMDs) for further translocation.…”

“…Of the three, metal ions with a tight first-coordination sphere closest to ideal octahedral geometry are the most stable (Zheng et al, 2017;Schmidt & Husted, 2019). Also, the coordinating environment is an important factor in deciding the physiological ligands for metal-binding SBPs (Vigouroux et al, 2020).…”

Iron Acquisition Systems of Gram-negative Bacterial Pathogens Define TonB-Dependent Pathways to Novel Antibiotics

“…According to this mechanism, the ABC-type transporter FpvDE ultimately transports ferrous iron into the cytoplasm. An overview of experiments concerning the fascinating path of pyoverdines from outside to inside the bacterial cell is found in Vigoroux et al 497 5.4.9. Mitochondrial Toxicity of Pvd.…”

Pseudomonas aeruginosa and its multiple strategies to access iron

“…After iron reduction, the ferrous iron is chelated by the periplasmic protein FpvC, which probably brings the metal to the FpvDE ABC transporter, allowing the passage of ferrous iron into the cytoplasm (Brillet et al, 2012). The structure of FpvC in complex with ferrous iron has been solved and shows an atypical coordination of ferrous iron, with an octahedral geometry involving six histidines, a unique example among iron‐binding proteins (Vigouroux et al, 2019). Native mass spectrometry approaches have shown that FpvF can bind apo‐pyoverdine and interact with the PvdRT‐OpmQ efflux system (Bonneau et al, 2020; Brillet et al, 2012).…”