Impact of the three-fold channel substitution D131N on kinetics of translocation of Fe2+ across the protein coat is more severe for human cytosolic H-chain ferritin than for human mitochondrial ferritin

Bugg, Zinnia, Hazlewood, Charlie, Hemmings, Andrew, Bradley, Justin and Le Brun, Nick (2026) Impact of the three-fold channel substitution D131N on kinetics of translocation of Fe2+ across the protein coat is more severe for human cytosolic H-chain ferritin than for human mitochondrial ferritin. Dalton Transactions. pp. 6844-6853. ISSN 1477-9226

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Abstract

Ferritins play a key role in iron management in organisms from all kingdoms of life. Excess iron is sequestered in mineral form within the hollow protein shell and can be liberated when supply becomes restricted. The protein consists of 24 isostructural monomeric units that pack with 4-, 3-, and 2-fold symmetry. Channels through the protein coat at the 3-fold axes of ferritins localised in the cytosol of animal cells contain a strictly conserved LCDFXEX ‘twin carboxylate’ motif, and have been shown to be the major iron entry route to animal ferritins, facilitating access to the H-chain intra-subunit catalytic ferroxidase centre. In the ferritin localised to the mitochondria of animals, there is natural variation within the residues lining this channel, such that the Asp residue of the twin carboxylate motif (Asp131) is not strictly conserved. Here we report X-ray crystallographic and solution kinetic studies of the properties of D131N variants of H-chain and mitochondrial ferritins. X-ray structures revealed significant perturbation of metal binding at the three-fold channels and ferroxidase centres of H-chain ferritin, but a relatively minor effect on mitochondrial ferritin. Likewise, kinetic data showed that rapid Fe2+ uptake was abolished in the D131N variant of H-chain ferritin, but less severely impacted in the equivalent variant of mitochondrial ferritin. Differences were also observed in rates of mineralisation and extent of iron release in the D131N variants of the two ferritins. The implications for the physiological role of mitochondrial versus cytosolic ferritin are discussed.

Item Type:	Article
Additional Information:	Data availability: Coordinate files detailing the structures derived from X-ray diffraction data can be downloaded from the protein data bank (https://www.rcsb.org) using the accession codes listed in Table S1 of the supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d5dt02739j. Other data for this article, including stopped-flow and standard kinetic data, and absorbance data, are available at OSF at https://doi.org/10.17605/OSF.IO/7GC8M.
Faculty \ School:	Faculty of Science > School of Chemistry, Pharmacy and Pharmacology Faculty of Science
UEA Research Groups:	Faculty of Science > Research Centres > Centre for Molecular and Structural Biochemistry Faculty of Science > Research Groups > Pharmacology and Biological Chemistry
Depositing User:	LivePure Connector
Date Deposited:	06 May 2026 09:03
Last Modified:	06 May 2026 09:03
URI:	https://ueaeprints.uea.ac.uk/id/eprint/102908
DOI:	10.1039/D5DT02739J

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