Disclosing the Molecular Mechanism of Iron Incorporation in Listeria innocua Dps by EPR Spectroscopy
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Applied Magnetic Resonance
ORIGINAL PAPER
Disclosing the Molecular Mechanism of Iron Incorporation in Listeria innocua Dps by EPR Spectroscopy Andrea Ilari1 · Giuliano Bellapadrona1 · Donatella Carbonera2 · Marilena Di Valentin2 Received: 2 July 2020 / Revised: 11 October 2020 / Accepted: 14 October 2020 © The Author(s) 2020
Abstract Bacteria overexpress, under condition of starvation or oxidative stress, Dps (DNAbinding 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 H 2O2 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. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s0072 3-020-01287-x) contains supplementary material, which is available to authorized users. * Andrea Ilari [email protected] * Marilena Di Valentin [email protected] 1
Institute of Molecular Biology and Pathology (IBPM), Consiglio Nazionale delle Ricerche (CNR), P.le A. Moro 5, Rome, Italy
2
Dipartimento di Scienze Chimiche, Università degli Studi di Padova, Via Marzolo 1, 35131 Padova, Italy
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1 Introduction Iron is essential for most living organisms since it serves as a cofactor in several enzymes and as a catalyst in electron transfer processes. However, iron is poorly available and potentially toxic, since Fe(III), the stable oxidation state of the metal at neutral pH values, forms insoluble hydroxy-aquo complexes, while Fe(II) reacts with oxygen forming reactive oxygen species (ROS) through the Fenton reaction:
Fe(II) + O2 → Fe(III) + O−2
(1)
2O−2 + 2H+ → H2 O2 + O2 ,
(2)
H2 O2 + Fe(II) → ⋅OH + OH− + Fe(III)
(3)
Hydroxyl radicals and hydrogen peroxide products in this reaction can damage both DNA, proteins and membrane lipids
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