BEORHN: Bacterial Enzymatic Oxidation of Reactive Hydroxylamine in Nitrification via Combined Structural Biology and Molecular Simulation

氮循环对环境和全球健康至关重要。现代农业中使用的大部分氮来自主要由氨或铵化合物组成的人造肥料。在土壤和水中的硝化细菌的作用下，它被转化为对植物有用的含氮化学品(例如硝酸盐)，然后通过进一步的细菌作用返回大气中的氮气。这些过程中的损失或失衡会导致污染物和温室气体一氧化二氮(N2O)、污染物一氧化氮(NO)、有毒中间体羟胺(NH2OH)或亚硝酸盐/硝酸盐释放到淡水中，导致藻类水华。因此，了解硝化过程对农业、粮食安全、环境和人类健康至关重要。在硝化过程中，第二步涉及活性化合物羟胺的氧化，由含有金属的蛋白质催化，这些蛋白质含有一种非常不寻常的铁-血红素结构，其中血红素含有一个额外的键或与蛋白质的“交联键”。羟胺氧化还原酶(HAO)和细胞色素P460(CytP460)这两个结构非常不同的蛋白质家族进行这种化学反应，产生不同的反应产物(HAO为NO，CytP460为N2O)。HAO的每个功能单元包含七个铁-血红素单元，这些单元起到转移或‘穿梭’电子的作用，以及一个p460血红素单元，在那里，血红素通过与酪氨酸氨基酸残基的交联进一步修饰，羟胺在那里发生氧化。在CytP460中，每个功能单元都包含一个催化P460单元，但在这种情况下，它与另一种氨基酸(赖氨酸)交叉连接。此外，增加复杂性的是，在CytP460家族中，到目前为止在不同细菌家族(欧洲支原体和胶囊支原体)中发现的两种蛋白质，尽管进行了完全相同的化学反应，但具有不同的血红素环境。我们的项目解决了硝化过程中知之甚少的第二步，即HAO和CytP460对羟胺的催化氧化。我们将通过结合集成的光谱和结构生物学方法以及使用高性能计算的计算化学来针对这些蛋白质系统。我们将使用X射线结晶学，几乎同时测量同一晶体的光谱数据，以指定正确的电子状态酶的活性部位。我们将使用数千个非常小的(微型)晶体在室温下获得酶的结构，并制作作用中的酶的结构电影(更传统的技术产生更类似于单个电影帧的平均结构)。这些光谱和结构数据将与最先进的计算方法(分子动力学和最近发展的量子力学/分子力学方法)相结合，以便在原子水平上更好地了解这些酶是如何工作的。通过这种方式将实验和模拟联系起来，我们将从根本上了解这些酶的功能，以及为什么它们催化的反应会产生不同的产品。我们的最终目标是设计新的突变酶，利用我们对它们的结构如何影响它们催化的反应的知识，将它们的产品从NO变成N2O，反之亦然，这样就展示了在未来的生物技术应用中控制催化的潜力。

The nitrogen cycle is critical to the environment and global health. The majority of nitrogen used in modern agriculture comes from artificial fertiliser comprised primarily of ammonia or ammonium compounds. This is converted into nitrogen-containing chemicals that are useful to plants (e.g. nitrate) by the action of nitrifying bacteria in soils and water and is then returned to nitrogen gas in the atmosphere through further bacterial action. Losses or imbalances in these processes lead to the release of the pollutant and greenhouse gas nitrous oxide (N2O), the pollutant nitric oxide (NO), the toxic intermediate hydroxylamine (NH2OH), or nitrites/nitrates into freshwater, resulting in algal blooms. Understanding the nitrification process is therefore critically important for agriculture, food security, the environment and human health. In the nitrification process, the second step involves the oxidation of the reactive compound hydroxylamine, catalysed by metal-containing proteins which contain a highly unusual iron-heme structure where the heme contains an additional bond or 'cross-link' to the protein. Two families of structurally very different proteins, hydroxylamine oxidoreductase (HAO) and cytochrome P460 (CytP460), carry out this chemical reaction to yield different reaction products (NO for HAO and N2O for CytP460). Each functional unit of HAO contains seven iron-heme units that function to transfer or 'shuttle' electrons and one P460 heme unit where the heme is further modified via cross-linking to a tyrosine amino acid residue and where the oxidation of hydroxylamine occurs. In CytP460s each functional unit contains one catalytic P460 unit but, in this case, cross linked to a different kind of amino acid (lysine). Furthermore, to add to the complexity, within the CytP460 family, the two proteins so far identified in different families of bacteria (N. europaea and M. capsulatus), have different heme environments despite carrying out exactly the same chemical reaction. Our project addresses this poorly understood second step in the nitrification process, namely the catalytic oxidation of hydroxylamine by HAO and CytP460. We will target these protein systems by combining integrated spectroscopic and structural biology approaches and computational chemistry using high performance computing. We will use X-ray crystallography with near-simultaneous measurement of spectroscopic data of the same crystal to assign correct electronic states to the enzyme's active site. We will use thousands of very small (micro)crystals to obtain structures of enzymes at room temperature and to produce structural movies of the enzymes in action (more traditional techniques produce an average structure more similar to a single movie frame). These spectroscopic and structural data will be combined with state-of-the-art computational methods (molecular dynamics and recently developed quantum mechanics/molecular mechanics approaches) to better understand at the atomic level how these enzymes work. Linking experiments and simulations in this way, we will obtain a fundamental understanding of the function of these enzymes, and why the reactions they catalyse result in different products. Our ultimate goal is to design new, mutated enzymes, using our knowledge of how their structure affects the reactions they catalyse, to change their products from NO to N2O and vice versa, so demonstrating the potential for control of catalysis in future biotechnological applications.