A new pathway for iron-sulfur cluster repair

Bacteria inhabit almost every environmental niche on Earth, including some that are so harsh that many other forms of life cannot survive. This success is at least in part due to the ability of bacteria to adapt to changes in environment, and this adaptation is rooted in their capacity to alter patterns of gene expression in response to external and internal cues. A key environmental parameter that is monitored by many bacteria is oxygen concentration. We are particularly interested in the bacterium Escherichia coli (E. coli). One of its remarkable properties is that it is able to thrive both in the presence and absence of oxygen. To do this it has to dramatically alter its metabolism, but this has consequences because without oxygen the potential for energy conservation and growth are limited compared to when oxygen is present. To test whether oxygen is present E. coli uses a protein called FNR, which acts as an oxygen sensor. It has a special co-factor called an iron-sulfur cluster that reacts with oxygen in a way that switches FNR off. This involves the conversion of the cluster from one form (called the [4Fe-4S] form) to another (called the [2Fe-2S] form). In the 'off-state' FNR is in its [2Fe-2S] form and cannot bind to DNA to activate expression of genes that are used during growth in the absence of oxygen. When there is no oxygen the iron-sulfur cluster remains in the [4Fe-4S] form and the protein can bind to DNA and activate expression of genes that are needed for growth in the absence of oxygen.
For the last few years we have studied the reaction of oxygen with the FNR iron-sulfur cluster. These studies have revealed the complex biochemistry of the reaction and how this makes the FNR protein an exquisitely sensitive oxygen sensor. We recently showed that in its 'off-state' the [2Fe-2S] cluster is different from most other previously characterised [2Fe-2S] cluster cofactors and that this is a result of the fact that it is formed through oxidative conversion (which can be thought of as oxidative damage) of the [4Fe-4S] form. This results in an unusual modification of the protein in which sulfide from the initial [4Fe-4S] cluster is attached to the protein during the conversion reaction. This can be thought of as a form of stored sulfur and, remarkably, this can be used to repair the cluster back to its original [4Fe-4S] form when oxygen is no longer present. The unusual [2Fe-2S] cluster of FNR has also been discovered in other iron-sulfur cluster proteins, raising the possibility that repair of oxidatively damaged [4Fe-4S] clusters via this mechanism might be widespread.
Using a wide range of approaches, we propose to investigate further the formation of the unusual [2Fe-2S] cluster and the repair of [4Fe-4S] cluster to determine the mechanism and the importance of this in vivo as a previously unrecognised pathway for repair of oxidatively damaged iron-sulfur clusters.

The bacterial transcriptional regulator FNR, which orchestrates the switch between aerobic and anaerobic respiration, becomes active under anaerobic conditions through coordination of a [4Fe-4S] cluster, leading to dimerization and DNA-binding. Oxygen triggers the conversion of the [4Fe-4S] cluster to a [2Fe-2S] form, causing the protein to monomerise with the loss of specific DNA-binding. Recently, we showed that cluster conversion leads to oxidation of the two cluster released sulfides and formation of Cys persulfides that coordinate the [2Fe-2S] cluster. This reaction is not unique to FNR, since it also occurs in radical-SAM enzymes. The [4Fe-4S] cluster in FNR can be repaired by anaerobic incubation with reductant and Fe(II) only, pointing towards a new mechanism for the assembly or repair of biological [4Fe-4S] clusters. Using a range of both in vivo and in vitro techniques, we propose to study three principal aspects: persulfide formation in FNR; FNR cluster repair; and, the generality of the cluster repair pathway.
Using mass spectrometry (MS), we will determine whether the formation of the persulfide occurs simultaneously with cluster conversion, or is a distinct step, also which Cys residues can incorporate sulfur to form persulfides, and how many oxygen molecules are required for persulfide formation. We will also detect persulfides formed in vivo by isolating FLAG-tagged protein from E. coli cultures. The in vivo importance of FNR cluster repair in the absence of de novo iron-sulfur cluster synthesis will be established using a variety of strategies. We will also determine the required properties of the reductant, and whether glutaredoxins can function in persulfide reduction. Finally, we will investigate whether the same cluster repair process occurs in other [4Fe-4S] cluster-containing proteins that are sensitive to oxidative damage and that have already been shown to generate Cys persulfides.

This project involves a fundamental study of the biochemistry and molecular biology of iron-sulfur cluster conversion reactions in response to oxygen/oxidative stress. 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:

- policy makers and commercial stakeholders, who are likely to be interested in this unusual iron-sulfur cluster repair pathway following oxidative damage, which is linked to range of diseases (e.g. Parkinson's and Alzheimer's) associated with ageing, and may see opportunities to develop new interventions for disease states in which damage to iron-sulfur clusters plays a significant role e.g. those associated with respiration, DNA replication and DNA repair. These groups will benefit from the high quality publications arising from this work, which will be accessible to researchers working in private (pharmaceutical) and public sector laboratories (e.g. health agencies), and by advisors to policy makers. This 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;

- the biotechnology and pharmaceutical sectors and public sector laboratories, from the point of view of benefiting from future employment of the state-of-the-art training in biochemistry, spectroscopy and molecular biology provided to PDRAs employed on the grant (and to PhD students and undergraduates working within the research groups who benefit from the expertise of the PDRAs);

- 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.