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Key carboxylate residues for iron transit through the prokaryotic ferritin SynFtn.

ABSTRACT: 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.

SUBMITTER: Bradley JM

PROVIDER: S-EPMC8743623 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cellular growth, function, and protection require proper iron management, and ferritin plays a crucial role as the major iron sequestration and storage protein. Ferritin is a 24 subunit spherical shell protein composed of both light (FTL) and heavy chain (FTH1) subunits, possessing complimentary iron-handling functions and forming three-fold and four-fold pores. Iron uptake through the three-fold pores is well-defined, but the unloading process somewhat less and generally focuses on lysosomal ferritin degradation although it may have an additional, energetically efficient pore mechanism. Hereditary Ferritinopathy (HF) or neuroferritinopathy is an autosomal dominant neurodegenerative disease caused by mutations in the FTL C-terminal sequence, which in turn cause disorder and unraveling at the four-fold pores allowing iron leakage and enhanced formation of toxic, improperly coordinated iron (ICI). Histopathologically, HF is characterized by iron deposition and formation of ferritin inclusion bodies (IBs) as the cells overexpress ferritin in an attempt to address iron accumulation while lacking the ability to clear ferritin and its aggregates. Overexpression and IB formation tax cells materially and energetically, i.e., their synthesis and disposal systems, and may hinder cellular transport and other spatially dependent functions. ICI causes cellular damage to proteins and lipids through reactive oxygen species (ROS) formation because of high levels of brain oxygen, reductants and metabolism, taxing cellular repair. Iron can cause protein aggregation both indirectly by ROS-induced protein modification and destabilization, and directly as with mutant ferritin through C-terminal bridging. Iron release and ferritin degradation are also linked to cellular misfunction through ferritinophagy, which can release sufficient iron to initiate the unique programmed cell death process ferroptosis causing ROS formation and lipid peroxidation. But IB buildup suggests suppressed ferritinophagy, with elevated iron from four-fold pore leakage together with ROS damage and stress leading to a long-term ferroptotic-like state in HF. Several of these processes have parallels in cell line and mouse models. This review addresses the roles of ferritin structure and function within the above-mentioned framework, as they relate to HF and associated disorders characterized by abnormal iron accumulation, protein aggregation, oxidative damage, and the resulting contributions to cumulative cellular stress and death.

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Key carboxylate residues for iron transit through the prokaryotic ferritin SynFtn.

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