Towards a complete structure-function description of the denitrification pathway

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The denitrification process is a four-step reduction of nitrate to dinitrogen via the intermediates nitrite (NO2) and the gaseous N-oxides NO and N2O. The loss of fixed nitrogen via these steps to inert dinitrogen is of major importance to the terrestrial and oceanic nitrogen cycles and has agronomic, environmental, and medical impacts. The current proposal builds on our recent exciting results on nitrite reductases from two dentrifiers, A. xylosoxidans and A. cycloclastes, and nitrous oxide reducatse (N2OR) from A. cycloclastes (our unpublished results). These enzymes catalyse the first committed step, namely the reduction of NO2, and the last step of the denitrification process. The project is aimed towards reaching a 'complete' understanding of the important steps of denitrification catalysed by these enzymes through combined structural, and enzymological studies and site directed mutations of these enzymes. Thus, for example, in the case of NiR, we propose to test different aspects of our proposed catalytic mechanism (PNAS, 23 Aug 2005) experimentally. Since the enzyme used in these studies was the green AcNiR, we wish to test the general occurrence of these intermediates in CuNiR turnover by extending this work to the blue AxNiR, for which we have some 20 potentially relevant mutant NiRs, many of which have been structurally characterized in their resting states. In the case of N2OR, on the basis of two structures we have obtained very recently, we have proposed a reaction mechanism which requires confirmation through an extensive experimental structure-function programme. Even though the recent structure determinations have advanced our knowledge in terms of the mechanism, they have also highlighted challenges in understanding how the difficult chemistry of N2O reduction is achieved by the novel CuZ cluster.