Routes of iron entry into, and exit from, the catalytic ferroxidase sites of the prokaryotic ferritin <i>Syn</i>Ftn

Bradley, Justin M.; Pullin, Jacob; Moore, Geoffrey R.; Svistunenko, Dimitri A.; Hemmings, Andrew M.; Brun, Nick E. Le

doi:10.1039/c9dt03570b

Cited by 11 publications

(21 citation statements)

References 27 publications

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“…Surprisingly, however, the rate at which the rapid iron oxidation phase of the apo protein was regenerated in variant D65A was also almost identical to that of the wild-type protein. Therefore, the impaired mineralization activity of variant D65A cannot be a consequence of increased stability of the di-Fe 3+ form of the FOC, as was reported for variant E62A [18], suggesting that the replacement of Asp65 has a less direct effect on the exit of Fe 3+ from the FOC.…”

Section: Resultsmentioning

confidence: 66%

“…The data reported here demonstrates that transport of iron to the FOC is inhibited in Syn Ftn variant E141A. However, the effect is less severe than that caused by disruption of the threefold channel by the D137A substitution that led to loss of all rapid Fe 2+ oxidation activity [18]. This suggests that residue Glu141 is not essential for Fe 2+ to traverse the protein coat, and that its role is to direct incoming metal toward the catalytic FOCs.…”

Section: Discussionmentioning

confidence: 88%

“…Firstly, substitution of Glu62 led to a reduction in the rate of rapid iron oxidation such that iron binding was no longer rate-limiting for this process [18]. No such effect was observed on substitution of Asp65.…”

Section: Discussionmentioning

confidence: 99%

“…The peptide sequence of Syn Ftn contains an N-terminal extension relative to other Ftns and this occludes the outer entrance of the B-channel (), suggesting a novel route of iron uptake in this protein. Indeed it was recently demonstrated that Fe 2+ enters Syn Ftn via the threefold channels [18] despite the absence of one of the carboxylates conserved in and essential to the equivalent channel in the animal ferritins. Furthermore, the conserved carboxylates of the transient binding sites of animal ferritins are absent from the Syn Ftn peptide sequence.…”

Section: Introductionmentioning

confidence: 99%

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Key carboxylate residues for iron transit through the prokaryotic ferritin SynFtn

et al. 2021

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Ferritins are proteins forming 24meric rhombic dodecahedral cages that play a key role in iron storage and detoxification in all cell types. Their function requires the transport of Fe2+ from the exterior of the protein to buried di-iron catalytic sites, known as ferroxidase centres, where Fe2+ is oxidized to form Fe3+-oxo precursors of the ferritin mineral core. The route of iron transit through animal ferritins is well understood: the Fe2+ substrate enters the protein via channels at the threefold axes and conserved carboxylates on the inner surface of the protein cage have been shown to contribute to transient binding sites that guide Fe2+ to the ferroxidase centres. The routes of iron transit through prokaryotic ferritins are less well studied but for some, at least, there is evidence that channels at the twofold axes are the major route for Fe2+ uptake. SynFtn, isolated from the cyanobacterium Synechococcus CC9311, is an atypical prokaryotic ferritin that was recently shown to take up Fe2+ via its threefold channels. However, the transfer site carboxylate residues conserved in animal ferritins are absent, meaning that the route taken from the site of iron entry into SynFtn to the catalytic centre is yet to be defined. Here, we report the use of a combination of site-directed mutagenesis, absorbance-monitored activity assays and protein crystallography to probe the effect of substitution of two residues potentially involved in this pathway. Both Glu141 and Asp65 play a role in guiding the Fe2+ substrate to the ferroxidase centre. In the absence of Asp65, routes for Fe2+ to, and Fe3+ exit from, the ferroxidase centre are affected resulting in inefficient formation of the mineral core. These observations further define the iron transit route in what may be the first characterized example of a new class of ferritins peculiar to cyanobacteria.

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Section: Resultsmentioning

confidence: 66%

Section: Discussionmentioning

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Key carboxylate residues for iron transit through the prokaryotic ferritin SynFtn

et al. 2021

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“…The proposal of dedicated routes for the transportation of Fe 2+ from bulk solution through the protein coat to the site of oxidation has faced resistance due to the existence of a channel directly linking the ferroxidase center to bulk solution (143). However, there is increasing evidence that networks of carboxylate residues with conformational flexibility play key roles in Fe 2+ transfer in all cage-forming ferritins (141,142,(144)(145)(146). All ferritins sequester Fe 2+ from solution and utilize an electron accepting co-substrate, such as O2 or H2O2, to drive its oxidation to the Fe 3+ state.…”

Section: Iron Storage In Bacteriamentioning

confidence: 99%

Bacterial iron detoxification at the molecular level

Bradley

Svistunenko

Wilson

et al. 2020

Journal of Biological Chemistry

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Iron is an essential micro-nutrient and, in the case of bacteria, its availability is commonly a growth-limiting factor. However, correct functioning of cells requires that the labile pool of chelatable ‘free’ iron is tightly regulated. Correct metalation of proteins requiring iron as a cofactor demands that such a readily accessible source of iron exists, but over-accumulation results in an oxidative burden that, if unchecked, would lead to cell death. The toxicity of iron stems from its potential to catalyze formation of reactive oxygen species (ROS) that, in addition to causing damage to biological molecules, can also lead to the formation of reactive nitrogen species (RNS). In order to avoid iron-mediated oxidative stress, bacteria utilize iron-dependent global regulators to sense the iron status of the cell and regulate the expression of proteins involved in the acquisition, storage and efflux of iron accordingly. Here, we survey the current understanding of the structure and mechanism of the important members of each of these classes of protein. Diversity in the details of iron homeostasis mechanisms reflect the differing nutritional stresses resulting from the wide variety of ecological niches that bacteria inhabit. However, in this review we seek to highlight the similarities of iron homeostasis between different bacteria, whilst acknowledging important variations. In this way we hope to illustrate how bacteria have evolved common approaches to overcome the dual problems of the insolubility and potential toxicity of iron.

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Disclosing the Molecular Mechanism of Iron Incorporation in Listeria innocua Dps by EPR Spectroscopy

et al. 2020

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Bacteria overexpress, under condition of starvation or oxidative stress, Dps (DNA-binding proteins from starved cells), hollow sphere formed by 12 identical subunits endowed with ferritin-like activity. The iron oxidation and incorporation in Dps take place using H2O2 produced under starvation as preferred iron oxidant, thereby protecting bacteria from oxidative damage. Even if the role of Dps is well known, the mechanism of iron oxidation and incorporation remain to be elucidated. Here, we have used the EPR technique to shed light on the Fe(II) binding and oxidation mechanism at the ferroxidase center using both the wild-type (wt) protein and mutants of the iron ligands (H31G, H43G and H31G-H43G-D58A). The EPR titration of wt Dps and the H31G mutant with Fe(II) upon H2O2 addition shows that Fe(II) is oxidized with the increase of the signal at g = 4.3, reaching a maximum for 12 Fe(II)/subunit. The EPR signal becomes negligible when the titration is carried out on the triple mutant. These experiments indicate that the iron firstly occupied the A site at the ferroxidase center and confirm that the residues H31, H43 and D58 have a key role in the iron oxidation and incorporation process. Moreover, the data indicate that the ferroxidase center, upon mutation of H31 or H43 to Gly, changes the mode of iron binding. Finally, we demonstrate here that, when the iron micelle forms, the EPR signal at g = 4.3 disappears indicating that iron leaves the ferroxidase center to reach the inner cavity.

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Routes of iron entry into, and exit from, the catalytic ferroxidase sites of the prokaryotic ferritin SynFtn

Abstract: This work describes the identification of two residues, D137 and E62, that are critical for, respectively, the transport of Fe2+ into, and Fe3+ out of, the catalytic sites of a prokaryotic ferritin.

Cited by 11 publications

References 27 publications

Key carboxylate residues for iron transit through the prokaryotic ferritin SynFtn

Key carboxylate residues for iron transit through the prokaryotic ferritin SynFtn

Bacterial iron detoxification at the molecular level

Disclosing the Molecular Mechanism of Iron Incorporation in Listeria innocua Dps by EPR Spectroscopy

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