About the Project
Nitrous oxide (N2O) is a greenhouse gas with a ~300 fold greater global warming potential than CO2 and an ability to deplete stratospheric ozone. Much of the N2O generated arises from activity of soil bacteria that are able to grow anaerobically through denitrification, a process central to the biogeochemical cycling of nitrogen, in which nitrate is reduced stepwise to dinitrogen (N2) via nitrite, nitric oxide (NO), and N2O. The last step of the pathway, reduction of N2O to the environmentally harmless N2, is catalysed by the enzyme nitrous oxide reductase (N2OR); failure of this step to occur is the major cause of N2O release into the atmosphere. Atmospheric levels of N2O are increasing, in large part due to the increased use in agriculture of nitrogenous fertilisers. N2OR is a soluble enzyme that contains two types of copper centre: CuA, a thiol-bridged dinuclear copper site; and CuZ, a unique tetranuclear Cu cluster that also contains sulfide. Assembly of the Cu cofactors of N2OR occurs in the periplasm and is dependent on a number of assembly proteins whose functions are, in most cases, not well defined.
This project aims to provide new molecular detail of the trafficking of copper and sulfide within the bacterial cell for assembly of Cu centres of N2OR, leading to new insights into the biogenesis of N2OR that will feed into long-term efforts towards maximising N2OR activity in soils, thereby reducing N2O emissions.
This multi-disciplinary project will be based in the Le Brun and Gates labs at UEA and the Balk lab at John Innes Centre and will involve microbiology, protein purification, biochemistry and bioanalytical techniques, offering excellent training potential for the student within a supportive and stimulating environment. Informal enquiries to Prof Nick Le Brun ([Email Address Removed]) are welcome.
i) Bennett, S., Torres, M., Soriano-Laguna, M., Richardson, D., Gates, A. J. & Le Brun, N. E. (2020) nosX is essential for whole cell N2O reduction in Paracoccus denitrificans but not for assembly of copper centres of nitrous oxide reductase. Microbiology. 166, 909-917
ii) Crack, J. C., Amara, P., Volbeda, A., Mouesca, J.-M., Rohac, R., Pellicer Martinez, M. T., Huang, C.-Y., Gigarel, O., Rinaldi, C.; Le Brun, N. E. and Fontecilla-Camps, J. C. (2020) Electron and proton transfers modulate DNA binding by the transcription regulator RsrR. J. Am. Chem. Soc. 142, 5104 – 5116
iii) Bennett, S. P., Soriano-Laguna, M. J., Bradley, J. M., Svistunenko, D. A., Richardson, D. J., Gates, A. J. and Le Brun, N. E. (2019) NosL is a dedicated copper chaperone for assembly of the CuZ center of nitrous oxide reductase. Chem. Sci. 10, 4985-4993
iv) Bradley, J. M., Svistunenko, D. A., Pullin, J., Hill, N., Stuart, R. K., Palenik, B., Wilson, M. T., Hemmings, A. M., Moore, G. R. and Le Brun, N. E. (2019) Reaction of O2 with a di-iron protein generates a mixed valent Fe2+/Fe3+ center and peroxide. Proc. Natl. Acad. Sci. U.S.A., 116, 2058-2067
v) Kay, K. L., Zhou, L., Tenori, L., Bradley, J. M., Singleton, C., Kihlken, M. A., Ciofi-Baffoni, S. and Le Brun, N. E. (2017) Kinetic analysis of copper transfer from a chaperone to its target protein mediated by complex formation. Chem. Comm. 53, 1397-1400