Nature's solution to the iron problem: Mechanisms of iron management in ferritins

Iron is essential for virtually all forms of life, playing central roles in many of the reactions on which life depends. Although this metal is highly abundant in the earth's crust, it is largely 'locked up' in minerals that are highly insoluble and this severely limits its availability to living organisms. The scarcity of iron has led to the evolution in microbes of many ingenious mechanisms to obtain sufficient quantities to sustain growth. These include the secretion and re-absorption of small organic molecules that can bind iron very tightly and therefore hoover up what iron there is. Pathogenic bacteria can effectively steal iron from iron-containing proteins in their hosts, and the success of such mechanisms is a key determinant of whether or not a successful infection is established. Another important aspect of how cells overcome the iron problem, is their ability to store iron. This is achieved through a remarkable family of proteins known as ferritins. These can be thought of as being football-like molecules with a hollow centre, inside which thousands of iron atoms can be stored in the form of an iron mineral - one that would form insoluble precipititates were it not for the solubilising effect of the protein coat. The storage of iron serves two important functions. Firstly, it enables microbes to draw on reserves when iron in the immediate environment becomes particularly 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. In this research we propose to better understand how two particular ferritins from two very different microbes take up iron and orchestrate the deposition of their iron mineral cargo, and how they subsequently release it. One of these is from the organism Escherichia coli - a bacterium that has for decades been a workhorse for understanding cellular processes, but which can also be pathogenic. The other ferritin is one that was very recently identified in phytoplankton. These organisms are responsible for a large proportion of the primary production of organic molecules from carbon dioxide in the oceans. Furthermore, they also form spectacular blooms in areas of temporary nutrient sufficiency. In many areas, the growth limiting nutrient is iron and it is now known that the presence of ferritin in these phytoplankton is crucial for their ability to utilise temporarily available iron. This research will lead to a significant advance in our understanding of iron cycling processes and of the relationship between structural and functional properties of ferritins. This will also have more general impact in understanding the reactivities of iron proteins and in protein-driven biomineralisation processes.

Iron is essential but presents organisms with a dual problem of poor bioavailability and toxicity. Part of Nature's solution to this problem is the ferritin family of proteins, which function in the storage and detoxification of iron and the release of iron for anabolic processes. Our recent work on bacterioferritin (BFR) from Escherichia coli has led us to propose a novel mechanism of iron mineralisation, in which the ferroxidase centre functions as a true catalytic cofactor, continually cycling between bridged di-Fe(III) (oxidised) and di-Fe(II) (reduced) states. Here, we will explore this mechanism in detail to determine how electrons resulting from the oxidation of Fe(II) in the cavity are channelled to the ferroxidase centre. We have already shown that a novel Fe(II)-binding site on the inner surface of the protein is important for this and we now need to determine the role of other residues in facilitating electron transfer. The structural differences associated with the catalytic centres of different ferritins clearly have significant consequences for their mechanistic properties. We will investigate how the structure of the ferroxidase centre controls its mechanistic properties, and discover if we can convert the BFR ferroxidase centre into one that functions as a eukaryotic H-chain-like centre. Under conditions of low iron, ferritin stores can be utilised to support growth and BFR may be important for supplying iron directly to the Suf iron-sulfur cluster biosynthesis pathway, which functions under conditions of iron starvation and oxidative stress. We will investigate this using a novel in vitro system. Recently it was shown that the ability of phytoplankton to form massive blooms in the ocean is associated with the ability to store and cycle growth-limiting iron through ferritin. We will investigate the mechanisms of iron mineralisation and release in this unusual eukaryotic ferritin.

The main beneficiaries of the proposed research will be the academic research community, but, as described in the beneficiaries section, this is potentially a broad group. Furthermore, the resulting increase in understanding of how iron is cycled in microbes could, in the longer term lead to applications in antibiotics and in biotechnology. For example, the possibility of disrupting iron release from ferritins could reduce the pathogenicity of at least some invading organisms. Furthermore, ferritins are increasingly being exploited for nanoscience applications (i.e. the generation of new nanoparticles) and the information we will gain here will undoubtedly impact on this. The connection between iron cycling in diatom phytoplankton and the conversion of carbon dioxide to organic molecules in the oceans also means that our research 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) initiative. We will evaluate the data that emerges from this work for potential commercial exploitation The vital role that iron and metal ions in general play in maintaining health (of e.g. humans, molluscs, yeast and bacteria) is really not well appreciated by the general public and the major beneficiaries of the research, in terms of appreciating this important area, are likely to be school children and the general public. A major focus of the impact of this research beyond academic researchers is to generate material appropriate for both Schools and for general public engagement to encourage the next generation to study science and in particular chemistry and biology, and to encourage a better appreciation of research generally. UEA has a well established infrastructure for schools and public outreach projects. Together with partner organizations such as Norwich City Council, Norfolk Museums Service, Eastern Daily Press, the BBC, and the BBSRC Institute of Food Research and John Innes Centre, it won a Beacon of Public Engagement award 'CueEast' (Community University Engagement East) in 2007, making it one of a handful of national public engagement coordinating centres. This provides an ideal environment for increasing impact of the research conducted at the University.