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N-氧化物NO和N2O将硝酸盐还原为氮素的四步反应。通过这些步骤将固定氮流失到惰性氮素，对陆地和海洋氮循环具有重要意义，并具有农学、环境和医学影响。目前的建议建立在我们最近关于两种树状菌(A.xylosoxidans和A.clclastes)的亚硝酸盐还原酶以及A.clclastes的一氧化二氮还原(N2OR)的令人兴奋的结果(我们的未发表结果)的基础上。这些酶催化了第一步，即NO2的还原，以及反硝化过程的最后一步。该项目的目的是通过对这些酶的结构、酶学和定点突变的结合研究，对这些酶催化的反硝化作用的重要步骤达成“完整”的了解。因此，例如，在NIR的情况下，我们建议通过实验测试我们提出的催化机理的不同方面(PNAS，2005年8月23日)。由于这些研究中使用的酶是绿色的AcNiR，我们希望通过将这一工作扩展到蓝色的AxNiR来测试这些中间体在CuNiR周转中的普遍存在，我们有大约20个潜在的相关突变体NIR，其中许多已经在其静止状态下被结构表征。在N2OR的情况下，在我们最近获得的两个结构的基础上，我们提出了一个需要通过广泛的实验结构-功能程序来证实的反应机理。尽管最近的结构测定在机理方面提高了我们的知识，但它们也突显了理解N2O还原的困难化学是如何通过新的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from two dentrifiers, A. xylosoxidans and A. cycloclastes, and nitrous oxide reducatse (N2OR) from A. cycloclastes (our unpublished results).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.