Towards a complete structure-function description of the denitrification pathway

反硝化过程是通过中间体亚硝酸盐（NO2）和气态氮氧化物NO和N2 O将硝酸盐还原为二氮的四步反应。通过这些步骤将固定氮损失为惰性二氮对陆地和海洋氮循环至关重要，并具有农学，环境和医学影响。目前的建议是建立在我们最近对来自两种dentrifiers，A。xylosoxidans和A. cycloclastes和N_2O还原酶（N_2OR）。cycloclastes（我们未发表的结果）。这些酶催化第一个关键步骤，即NO2的还原，以及反硝化过程的最后一步。该项目旨在通过结合结构和酶学研究以及这些酶的定点突变，对这些酶催化的反硝化作用的重要步骤有一个“完整”的理解。因此，例如，在NiR的情况下，我们建议实验测试我们提出的催化机制的不同方面（PNAS，2005年8月23日）。由于这些研究中使用的酶是绿色AcNiR，我们希望通过将这项工作扩展到蓝色AxNiR来测试CuNiR周转中这些中间体的一般发生，为此我们有大约20个潜在相关的突变NiR，其中许多在其静息状态下具有结构特征。在N2 OR的情况下，根据我们最近获得的两种结构，我们提出了一种反应机制，需要通过广泛的实验结构-功能程序进行确认。尽管最近的结构测定在机制方面推进了我们的知识，但它们也突出了理解N2 O还原的困难化学如何通过新型CuZ簇实现的挑战。

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two dentrifiers, A. xylosoxidans and A. cycloclastes, and nitrous oxide reducatse (N2OR) from A. cycloclastes (our unpublished results).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