Towards a complete structure-function description of the denitrification pathway

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from two dentrifiers, A. xylosoxidans and A. cycloclastes, and nitrous oxide reducatse (N2OR) from A. cycloclastes (our unpublished results).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.

Our work has led to structure-based catalytic mechanisms for the enzymes NiR (AxNiR and AcNiR) and N2OR (AcN2OR), which perform the first committed and final steps in the bacterial denitrification process. We are in a strong position to test these mechanisms via an extensive structure-function and site-directed mutagenesis programme. 1. AcNiR: We used crystal harvesting to isolate and determine structures of several novel stable species with bound nitrite or NO, or both. The material for this study was purified by crystallisation on a small scale. We will purify larger quantities of the nitrosyl AcNiR complex by chromatographic methods and also generate the complex from resting-state NiR by inducing enzyme turnover in the presence of limiting substrate. We will then test the reactivities of the complex with excess nitrite or NO using EPR spectroscopy and gas chromatography. This will provide a crucial test of the proposed mechanism. 2. AxNiR: We propose to: (i) isolate the nitrosyl and NO2 complexes described above for AcNiR in AxNiR to demonstrate the universality of our proposed mechanism. (ii) the orientation of nitrite in mutants of green AfNiR is found to be correlated with catalytic activity. Using the large number of mutants available for AxNiR, we will determine the nitrite-soaked structures of these in order to test this hypothesis. (iii) produce new mutants of AxNiR with the aim of defining the proton pathway/delivery, which remains the least understood component of the mechanism. 3. N2OR: We have proposed a catalytic mechanism using our recently solved crystal structure of AcN2OR in two forms. (i). We will extend the resolution of these crystals and determine the N2O and ligand bound structures. (ii). We are in the final stages of expressing the gene encoding AxN2OR, thus a number of mutants can be produced readily to address: (a) electron transfer between the CuA and CuZ sites, (b) substrate binding and (c) the proton pathway.