A new pathway for iron-sulfur cluster repair

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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.