Abstract
Siderophores make iron accessible under iron-limited conditions and play a crucial role in the survival of microorganisms. Because of their remarkable metal-scavenging properties and ease in crossing cellular envelopes, siderophores hold great potential in biotechnological applications, raising the need for a deeper knowledge of the molecular mechanisms underpinning the siderophore pathway. Here, we report the structural and functional characterization of a siderophore-interacting protein from the marine bacterium Shewanella frigidimarina NCIBM400 (SfSIP). SfSIP is a flavin-containing ferric-siderophore reductase with FAD- and NAD(P)H-binding domains that have high homology with other characterized SIPs. However, we found here that it mechanistically departs from what has been described for this family of proteins. Unlike other FAD-containing SIPs, SfSIP did not discriminate between NADH and NADPH. Furthermore, SfSIP required the presence of the Fe2+-scavenger, ferrozine, to use NAD(P)H to drive the reduction of Shewanella-produced hydroxamate ferric-siderophores. Additionally, this is the first SIP reported that also uses a ferredoxin as electron donor, and in contrast to NAD(P)H, its utilization did not require the mediation of ferrozine, and electron transfer occurred at fast rates. Finally, FAD oxidation was thermodynamically coupled to deprotonation at physiological pH values, enhancing the solubility of ferrous iron. On the basis of these results and the location of the SfSIP gene downstream of a sequence for putative binding of aerobic respiration control protein A (ArcA), we propose that SfSIP contributes an additional layer of regulation that maintains cellular iron homeostasis according to environmental cues of oxygen availability and cellular iron demand.
Highlights
Siderophores make iron accessible under iron-limited conditions and play a crucial role in the survival of microorganisms
Various Shewanella strains were identified as capable producers of soluble or surface-associated metal chelating compounds designated as siderophores
N-terminal sequence analysis of the purified protein confirmed the identity of SfSIP and the UV-visible spectrum presented the typical features of an oxidized flavoprotein with absorption peaks at 387 and 470 nm [23]
Summary
Siderophores make iron accessible under iron-limited conditions and play a crucial role in the survival of microorganisms. Various Shewanella strains were identified as capable producers of soluble or surface-associated metal chelating compounds designated as siderophores (from the Greek “iron carrier”) This is a strategy of iron uptake that is widespread across the tree of life, but poorly explored in the Shewanella genus [12, 13]. SIPs can be distinguished in two broad groups: one that generates cytosolic reduced free flavins that subsequently reduce the ferric-siderophores, and a second group of SIPs that contain stably attached flavins and use NADH and/or NADPH as reducing agents The latter includes the first reported SIP, the ViuB from Vibrio cholerae, and other examples such as Escherichia coli YqjH and FscN from the Gram-positive bacterium Thermobifida fusca (16 – 20).
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