Mechanistic studies of mitochondrial ferritin, a key player in iron mediated oxidative stress response and cellular iron metabolism

Iron is essential for virtually all forms of life, playing central roles in many of the reactions on which life depends. In animals, including humans, many of the iron-containing cofactors that are essential are synthesized in a cellular organelle called the mitochondrion, which consequently has a high requirement for iron and can be regarded as the major cellular hub for iron metabolism. Despite its importance, we currently have a rather incomplete picture of how iron is regulated in mitochondria. An important player in this is mitochondrial ferritin, a member of the remarkable ferritin family of proteins. Ferritins can be thought of as being football-like molecules with a hollow centre in which thousands of iron atoms can be stored in the form of an iron mineral - one that would form insoluble precipitates were it not for the solubilising effect of the protein coat. The storage of iron serves two important functions. Firstly, it enables cells to draw on reserves when iron is low, and secondly, it overcomes the potential toxicity of iron that results from the very properties that make it useful to life: without proper control, iron can lead to the generation of reactive oxygen species that can cause severe cellular damage. The function of mitochondrial ferritin appears to be principally one of protecting against iron toxicity. Accordingly, it is only expressed in tissues that are metabolically highly active with a high turnover of oxygen, and consequently particularly susceptible to oxidative stress. These tissues include heart, testis and brain neurones, and it has been shown that this ferritin protects mitochondria in these cells from oxidative stress. It is presumed that it does this through the sequestration of potentially harmful excess free iron, but the details of this process are unclear. Mitochondrial ferritin also participates in the regulation of iron distribution between different cellular compartments, with increased levels of the protein associated with low cytosolic iron and cytosolic ferritin levels. The reason for this is currently not clear. Finally, mitochondrial ferritin is involved in pathogenesis of neurodegenerative diseases; its expression levels are increased in a range of disorders, including Parkinson's diseases, Alzheimer's disease, restless legs syndrome and Friedreich's ataxia, but its connection to these diseases is unclear.
While studies of the in vivo roles of mitochondrial ferritin are increasing in scope and breadth, understanding of its mechanistic properties - how it interacts with iron and fulfils its cellular function - are lagging behind. Here, we propose a programme of research in which we will apply state of the art methodologies that will lead to important new information about how mitochondrial ferritin binds, oxidises and stores iron, and hence minimises its toxicity. Our preliminary studies have revealed that iron oxidation in mitochondrial ferritin involves protein-based radical formation. We will determine the role that the radical plays in mitochondrial ferritin and, in doing so, we will discover how it prevents the formation of reactive oxygen species that cause oxidative stress. We will also investigate iron release from mitochondrial ferritin, determining whether sequestered iron can be recycled. This will provide important new information about why the expression of mitochondrial ferritin causes iron deficiency in other cellular compartments.

As the site of biosynthesis for much of the cell's iron-sulfur clusters and heme, mitochondria have a high requirement for iron, but how this is regulated in the cell is not well understood. In tissues that are highly metabolically active, including heart and brain (neurones), mitochondria contain an unusual ferritin (FtMt), which has been shown to protect against the oxidative stress that inevitably results from the high turnover of iron and oxygen. Increased levels of FtMt is associated with low cytosolic iron/cytosolic ferritin and increased transferrin receptor levels, consistent with the idea that iron delivery to mitochondria is prioritised. There have been very few biochemical studies of FtMt (compared to the intense research effort on cytosolic H-chain ferritins) and information about the mechanism by which this protein oxidises and detoxifies iron is lacking. Previous studies indicated that it exhibits unusual behaviour that is both significantly different from cytosolic H-chain and at least in part unexplained. Furthermore, its iron release properties are entirely unexplored. In this application we propose to elucidate the mechanism of iron oxidation/mineralisation/detoxification and release in FtMt. We will use time-resolved UV-visible, EPR and Mössbauer spectroscopies to follow metal oxidation state changes as FtMt encounters Fe2+ in the presence of O2, and to detect the formation and decay of a Tyr radical species that is likely to have key functional importance. Solution kinetic studies and time-resolved X-ray crystallography will also be applied to wild type and site directed variants of FtMt to study both iron uptake and release. These biophysical approaches, together with cellular studies of oxidative stress protection conferred by site-directed variants of FtMt, will enable essential progress in understanding the biochemistry of FtMt to be made.

This project involves a fundamental study of the biochemistry of mitochondrial ferritin, a key player in iron management in the cell's major iron-utilising organelle. The project will have diverse and far reaching impacts within the UK and internationally. Outside of academia, there are several groups of potential beneficiaries, including:
- the healthcare sector. Although this is fundamental research, it holds promise to provide much needed detailed mechanistic information about the iron detoxifying function of mitochondrial ferritin, and whether the protein functions as a dynamic iron store, or simply as a sink for iron. These data will feed into studies of healthy ageing and of neurodegenerative diseases in which iron build up in neurones is known to occur and to be linked to the pathology of these diseases;
- health related policy makers and commercial stakeholders, who will be interested in the anticipated advances in understanding of iron biochemistry in the mitochondria of metabolically highly active cells, from the perspective of healthy ageing and understanding neurological disorders that have a huge social and economic impact in the UK and internationally;
- the biotechnology and pharmaceutical sectors. The work will contribute to the growing understanding of the rich variety of iron-oxygen reactivities of ferritins that could lead to applications in biocatalysis and synthetic biology. This is particularly so because some of the chemistry we have recently discovered is unprecedented amongst known iron proteins, and therefore will attract wide attention. Ferritins are increasingly being exploited for nanoscience applications (e.g. the generation of new nanocages for drug delivery or as nanoreactors) and the information we will gain here will undoubtedly impact on this;
- the environmental sustainability sector. The connection between iron cycling in diatom phytoplankton/cyanobacteria and the conversion of carbon dioxide to organic molecules in the oceans also means that our research on ferritins in general will impact on the field of geochemical cycling. This is a major research theme across the UEA and relates directly to the UEA-John Innes Centre ELSA (Earth and Life Systems Alliance) research initiative;
- the biotechnology and pharmaceutical sectors and public sector laboratories, from the point of view of benefiting (in terms of future employment) from the state-of-the-art training in biochemistry, spectroscopy and X-ray crystallography provided to the PDRA and to PhD students and undergraduates working within the research groups who benefit from the expertise of the PDRA;
- schools and the general public, who benefit from engagement activities running parallel with the research effort, which seek to inspire the next generation of science undergraduates and scientists and to better inform the general public of key scientific concepts and issues over which society has an influence. The vital role that iron, and metal ions in general, play in maintaining health is not well appreciated by the general public. Proteins that bind metal cofactors account for at least 30% of all proteins, and as such constitute a very important subgroup. The PI has a lot of experience of delivering engaging presentations in particular to A-level students.
The high quality publications arising from this work will be widely accessible to other researchers and advisers to policy makers and will stimulate new research and inform decision making. Although the project involves basic research, both Universities have appropriate policies and support (including training sessions) to identify any commercial opportunities arising from research activities and mechanisms to ensure that potential beneficiaries and investors are informed. The applicants are keen to exploit any commercial opportunities, although it is recognised that these are likely to arise in the longer term.