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

点击翻译按钮获取中文摘要

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.