Iron Uptake Oxidoreductase (IruO) Uses a Flavin Adenine Dinucleotide Semiquinone Intermediate for Iron-Siderophore Reduction

Abstract

Many pathogenic bacteria including Staphylococcus aureus use iron-chelating siderophores to acquire iron. Iron uptake oxidoreductase (IruO), a flavin adenine dinucleotide (FAD)-containing nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductase from S. aureus, functions as a reductase for IsdG and IsdI, two paralogous heme degrading enzymes. Also, the gene encoding for IruO was shown to be required for growth of S. aureus on hydroxamate siderophores as a sole iron source. Here, we show that IruO binds the hydroxamate-type siderophores desferrioxamine B and ferrichrome A with low micromolar affinity and in the presence of NADPH, Fe(II) was released. Steady-state kinetics of Fe(II) release provides k/K values in the range of 600 to 7000 M s for these siderophores supporting a role for IruO as a siderophore reductase in iron utilization. Crystal structures of IruO were solved in two distinct conformational states mediated by the formation of an intramolecular disulfide bond. A putative siderophore binding site was identified adjacent to the FAD cofactor. This site is partly occluded in the oxidized IruO structure consistent with this form being less active than reduced IruO. This reduction in activity could have a physiological role to limit iron release under oxidative stress conditions. Visible spectroscopy of anaerobically reduced IruO showed that the reaction proceeds by a single electron transfer mechanism through an FAD semiquinone intermediate. From the data, a model for single electron siderophore reduction by IruO using NADPH is described.

Structure and reactivity of a siderophore-interacting protein from the marine bacterium Shewanella reveals unanticipated functional versatility

“…The SIP superfamily, which includes both SIPs and FSRs is widespread across several bacterial genera, however, very few of these proteins have been thoroughly characterized (YqhJ from E. coli, FscN from T. fusca, and Iruo from Staphylococcus aureus) and thus, the specific intracellular fate of the iron-loaded siderophores is among the least understood steps of the siderophore pathway at the molecular level (16,17,21). Across the different genomes in the Shewanella genus, some species contain genes that code for SIPs only (e.g.…”

“…The electrode was then immersed in 2 ml of a mixed buffer solution containing 5 mM HEPES, 5 mM CHES, and 5 mM MES at the following pH values: 5.5, 6, 7.8, and 8.5. For each pH, voltammograms were acquired at the following scan rates (mV s Ϫ1 ): 10,13,17,21,28,36,41,48,60,78,100,130,170,210,280,360,410,480, 600, 780, 1,000, 1,300, 1,700, 2,100, 2,800, 3,600, 4,100, 4,800, 6,000, 7800, 9,100, and 10,000 to ensure uniform data point coverage in a logarithmic scale plot. After each experiment, the solution was removed, and the pH was measured.…”

Overview on the Bacterial Iron-Riboflavin Metabolic Axis

“…In Staphylococcus aureus , FAD functions as cofactor for IruO, a reductase of IsdG and IsdI, which are enzymes required for the degradation of heme. IruO is also directly required for iron reduction during its release from siderophores ( Kobylarz et al, 2017 ). In addition, two more other bacterial reductases required for iron release from siderophores depend upon FAD as cofactor, YqjH in E. coli ( Bamford et al, 2008 ) and FscN in Thermobifida fusca ( Li et al, 2015 ).…”

A detailed in silico analysis of secondary metabolite biosynthesis clusters in the genome of the broad host range plant pathogenic fungus Sclerotinia sclerotiorum

“…Genes in both intracellular and extracellular siderophore clusters were downregulated in planta relative to in vitro, with the exception of homologues of NRPS2 in the B. cinerea intracellular siderophore cluster (upregulated at all time points > 1 HPI) and an FAD-linked oxidoreductase upregulated at 24 and 48 HPI. The oxidoreductase may have a role in decoupling iron from the siderophore complex so that it can be used in the cell, as observed in the bacterium Staphylococcus aureus [59]. Genes in the extracellular siderophore cluster reached their minimum expression levels at 12 HPI, before increasing from 12 to 24 HPI.…”