Skip to main content

Kinetic studies on the reduction of dinitrogen by nitrogenase exploiting thealtered reactivity of heterologous enzyme systems

Objective



Research objectives and content:
The proposed project addresses questions which are central to our understanding of how N2 is reduced by nitrogenase, the enzyme system responsible for biological nitrogen fixation. Although the 3 dimensional structures of the nitrogenase proteins are known, that of the MoFe is in an oxidation state incapable of binding N2. Theoretical modelling studies suggest that the substrate-binding site must open up to allow binding and reduction of N2. To test this hypothesis, the mixed species enzyme formed between components of Klebsiella pneumoniae and Clostridium pasteurianum nitrogenases will be investigated. This system shows a time-dependent change in substrate specificity, showing no lag for the reduction of protons to hydrogen, but 500s and 1800s lags before acetylene and nitrogen become effective substrates. This system provides and opportunity to identify spectroscopic species and enzyme-bound intermediates involved in the reduction of different substrates. In particular, it should allow changes in the structure of the active site, proposed to allow the binding of N2, to be examined.Training content (objective, benefit and expected impact)
The training associated with this proposal, i.e. the techniques of anaerobic protein purification and manipulation of samples, steady-state kinetic analysis, EPR, ENDOR, FTIR and Mo X-ray absorption spectoscopies, together with chemical quench methods will applied to this problem. The benefit of such training, which will be given by acknowledged experts in these techniques, will be to broaden my range of skills which will then be applicable to a wide range of other systems of bioinorganic importance. Links with industry / industrial relevance (22)
None.

Funding Scheme

RGI - Research grants (individual fellowships)

Coordinator

John Innes Centre
Address
Norwich Research Park Colney
NR4 7UH Norwich
United Kingdom