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IruO Uses an FAD Semiquinone Intermediate for Iron-Siderophore Reduction Kobylarz, Marek J.; Heieis, Graham; Loutet, Slade A.; Murphy, Michael E. P.
Abstract
Many pathogenic bacteria including Staphylococcus aureus use iron-chelating siderophores to acquire iron. IruO, an FAD-containing 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 kcat/Km values in the range of 600 to 7000 M-¹s-¹ 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.
Item Metadata
Title |
IruO Uses an FAD Semiquinone Intermediate for Iron-Siderophore Reduction
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Creator | |
Contributor | |
Publisher |
American Chemical Society
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Date Issued |
2017-05-02
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Description |
Many pathogenic bacteria including Staphylococcus aureus use iron-chelating siderophores to
acquire iron. IruO, an FAD-containing 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 kcat/Km
values in the range of 600 to 7000 M-¹s-¹ 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.
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Subject | |
Genre | |
Type | |
Language |
eng
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Date Available |
2021-04-07
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Provider |
Vancouver : University of British Columbia Library
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Rights |
Attribution-NonCommercial-NoDerivatives 4.0 International
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DOI |
10.14288/1.0396559
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URI | |
Affiliation | |
Citation |
Kobylarz, M.J., Heieis, G.A., Loutet, S.A. & Murphy, M.E.P. (2017) Iron uptake oxidoreductase (IruO) uses a flavin adenine dinucleotide semiquinone intermediate for iron-siderophore reduction. ACS Chem. & Biol. 12: 1778-86.
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Publisher DOI |
10.1021/acschembio.7b00203
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Peer Review Status |
Reviewed
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Scholarly Level |
Faculty; Postdoctoral
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Rights URI | |
Aggregated Source Repository |
DSpace
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Item Citations and Data
Rights
Attribution-NonCommercial-NoDerivatives 4.0 International