Biological roles and mechanisms of nitric oxide reactions with iron-sulfur cluster transcriptional regulators

Nitric oxide is a poisonous molecule that is generated by soil bacteria and in our bodies as a defence against pathogenic organisms trying to establish infection. One of the major ways by which nitric oxide exerts its toxic effects is through reaction with a widespread group of proteins that contain a type of cofactor made from both iron and sulfur (called an iron-sulfur cluster). Members of this group play crucial roles in a very wide range of cellular processes. To avoid nitric oxide toxicity, disease-causing (as well as benign) bacteria have evolved protective systems that function to detoxify nitric oxide by removing it through chemical reaction. The fact that iron-sulfur cofactors are particularly sensitive to nitric oxide has been exploited in Nature, through the evolution of a number of regulatory proteins that themselves contain an iron-sulfur cluster and which function as biological switches, turning on the cellular nitric oxide detoxification response in the presence of nitric oxide. Despite the importance and widespread nature of the reaction of iron-sulfur clusters with NO, very little is known about this reaction process. This application is focussed on understanding how NO-responsive iron-sulfur cluster-containing regulators function. Here, we propose to investigate two such regulators (called WhiD and NsrR). One (WhiD) is a member of a family of proteins that are found only in a small number of bacteria (including Mycobacteria tuberculosis, the causative agent of tuberculosis, one of the world's major killers, and Streptomyces coelicolor, the source of many of the antibiotics currently in use in the clinic). Members of this protein family are known to play key roles in these bacteria in cell developmental processes associated with stress response, and are crucial for the ability of M. tuberculosis to survive in the inhospitable environment of a human host for years, in a dormant state that is highly resistant to antibiotics. The other (NsrR), is a member of a widely distributed but largely unstudied family of regulators. It functions as a primary NO sensor by controlling the cellular response to NO toxicity. Recent work in our laboratories has revealed important new insight into the nature of these regulatory proteins, including, for the first time, detailed mechanistic information about the reaction of a protein-bound iron-sulfur cluster with nitric oxide, leading to the formation of previously unreported products. We now propose to exploit these recent advances to explore, using a wide range of methods, the biochemistry of the reaction of NO with these proteins. This will reveal unprecedented mechanistic insight into how NO-sensing regulatory proteins function, and provide information that will be of general importance for all iron-sulfur protein NO reactions.

The ability to sense and respond to NO is important for the survival and adaptability of many bacteria. One of the major ways by which nitric oxide exerts its toxic effects is through reaction with iron-sulfur cluster-containing proteins, which are found in all cell types, playing crucial roles in a very wide range of processes, including respiration and protein synthesis. The particular sensitivity of iron-sulfur clusters to NO has been exploited in Nature: several NO-responsive regulators are themselves iron-sulfur cluster proteins. This application is focussed on understanding how the iron-sulfur clusters of regulatory proteins sense NO, and we propose to achieve this by elucidating the mechanisms by which clusters react with NO, what the iron-nitrosyl products are and determining the effect of this is on the DNA-binding characteristics of the regulator. We propose to study two NO-responsive regulators from S. coelicolor with which we have made important recent progress: NsrR, a member of the poorly studied Rrf2 family, regulates NO-detoxification systems that function to remove NO by redox reaction and is found in a wide range of pathogenic and non-pathogenic bacteria; WhiD is a member of the WhiB-like (Wbl) family of regulators (found only in the actinomycetes, which includes mycobacteria and Streptomyces), which play key roles in cell developmental processes such as sporulation. Our recent work on these two regulators has established them as ideal model systems, and we are now in a position to make important advances. We propose to use spectroscopic, kinetic and bioanalytical methods to reveal details about the cluster environments, the mechanism of reaction with NO and the products of cluster nitrosylation. For WhiD, we have shown that these are not the widely reported DNIC species. We will also investigate how reaction with NO affects DNA-binding. These studies will provide novel insight that will be important for understanding NO toxicity in general.

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. These fundamental studies have already provided a major breakthrough in understanding iron-sulfur cluster sensitivity to nitric oxide, and the work proposed here will exploit this head start to maximum effect. In the longer term, the detailed knowledge about WhiD/NsrR and other Wbl/Rrf2 family members gained as a result of this work may be exploited. Bacterial pathogens that cannot sense and respond to nitric oxide have decreased fitness or are unable to survive inside the host. Clearly, compounds that interfere with the NO sensing mechanisms of NsrR/WhiD could find widespread use as antibacterial drugs. The work outlined in this proposal will lay the groundwork for future structural studies which would be the first step in developing inhibitors of these sensing pathways. We will evaluate the data that emerges from this work for potential commercial exploitation. The vital role that iron, including iron-sulfur clusters, and metal ions in general play in maintaining health (in all living organisms) 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. We will present our work, at the appropriate level, as we have done often in the recent past, at outreach events for both the general public and particularly high school students to encourage the next generation to study science and in particular chemistry and biology, and to encourage a better appreciation of research in general. In this way, we will ensure impact of this research beyond academia. 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.