Award details

Enzymes and proteins of the Nitrogen Cycle

Reference	BBS/E/J/00000203
Principal Investigator / Supervisor	Dr Robert Eady
Co-Investigators / Co-Supervisors
Institution	John Innes Centre
Department	John Innes Centre Department
Funding type	Research
Value (£)	951,272
Status	Completed
Type	Institute Project
Start date	01/04/1997
End date	17/12/2003
Duration	80 months

Abstract

This project combines studies of nitrogenase, the enzyme responsible for biological nitrogen fixation, and enzymes and ancillary proteins involved in denitrification. These processes, which are restricted to microorganisms, are two important steps of the Nitrogen Cycle controlling the availability of fixed N to higher organisms. We study components of the denitrification pathway (the stepwise conversion of nitrate via nitrite to form NO, N2O and N2) in the soil microbe Alcaligenes xylosoxidans. A range of spectroscopic and kinetic techniques, combined with mutagenesis and X-ray crystallography, is focussed on analysis of the structure/function of the dissimilatory nitrite reductase (NiR). Our structural studies have shown an associated protein, cytochrome c', has the novel ability to discriminate between NO and CO, which bind to opposite faces of the heme. Resonance Raman and transient kinetics and time-resolved FTIR are combined with mutagenesis approaches to understand how this is controlled. Structure/function studies of N2O reductase, which converts this greenhouse gas to dinitrogen, and azurin as electron donor to NiR, are also under investigation. Nitrogenase catalysis involves the integration of ATP-driven proton and electron transfer to effect the binding and reduction of N2 to ammonia. Understanding the mechanism of nitrogenase at an atomic level is key to harnessing its chemistry. It is also a model for a variety of energy transduction processes involving electron transfer and alternate protein conformations coupled to nucleotide binding/hydrolysis. We study this aspect of nitrogenase function from the structure and reactivity of stable complexes of the two component proteins of nitrogenase (Mg.ATP-bound, MgADP.Pi-bound and heterologous enzymes) using small angle x-ray scattering, EPR, fluorescence spectroscopy and transient kinetics.

Summary

unavailable

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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