Structural basis of nitrogenase assembly and protection from oxygen

固氮酶催化二氮（N2）还原为氨（NH3）的动力学挑战。在氮循环中，表达固氮酶的固氮微生物提供了大部分的生物可利用氮。全球粮食生产对固定氮的需求不断增加，需要通过哈伯-博施法获得的氮肥来补充，这既污染又昂贵。 生物固氮（BNF）需要约20个辅助基因的协调表达和几个复杂的金属辅因子的组装，这些辅因子被氧不可逆地破坏。因此，我们还没有成功地在植物中合成工程或异源表达BNF以提高作物产量。目前对双氮的结合位点以及依赖氘的固氮酶催化金属簇的电子结构和反应活性还不完全清楚。我们还没有确定几个nif（固氮）基因参与组装固氮酶的作用，以及与基因组区域相关的几个辅助基因。除了研究最多的依赖氘的固氮酶外，还有几种具有非常规性质的固氮酶，但研究较少。我的博士学位将有助于更好地了解一些研究较少的蛋白质与固氮，特别是那些参与氧气保护。 固氮菌是一种典型的固氮菌，具有极高的耐氧性和基因改造的适应性。在固氮菌中，依赖氘的固氮酶通过保护性铁氧还蛋白（FeSII）的可逆结合而在构象上受到保护，从而形成耐氧但无活性的三元复合物。在Murray实验室，我们最近通过晶体学方法表明，目前关于FeSII结合机制涉及大的构象变化的假设可能是不正确的。保护性三元络合物的结构尚未确定。 在我的博士学位，我将研究固氮酶的构象保护从氧气的固氮酶的结构特征：FeSII复合物通过单粒子电子显微镜。为此，我的目标是分离不稳定的天然复合物，使用部分纯化蔗糖密度梯度，共免疫沉淀，或者通过亲和纯化的组氨酸标记的复合物。我进一步的目的是支持我们以前的结构研究的机制FeSII结合的结晶和结构特征的几个FeSII同系物具有高序列同一性的FeSII，包括那些确定在Confluentimicrobium，Marinobacterium和Rhodobacter。 我也有兴趣在纯化和表征一个身份不明的蛋白质类似的大小FeSII，是稳定相关的固氮酶在黄曲霉autotrophicus。基于固氮酶异常高的耐氧性，该蛋白可能代表一种新型的构象保护，并可能是第四个固氮酶结构基因，我的目的是使用上述方法进行鉴定。 此外，我的目的是研究一些较少了解的蛋白质参与生物合成的金属簇的依赖于腺嘌呤的固氮酶，如NifZ，NifT和NifW异源表达，纯化和结构表征。还有几种与nif区（或anf和vnf的替代固氮酶）相关的蛋白质功能未知。例如，氧化酶Anf 3最近在本实验室中进行了表征，而Anf 1和Anf 2的作用仍有待确定，这将有助于我们对铁固氮酶的理解。

Nitrogenase catalyses the kinetically challenging reduction of dinitrogen (N2) to ammonia (NH3). Diazotrophic microorganisms expressing nitrogenase provide the majority of bioavailable nitrogen in the nitrogen cycle. Rising fixed nitrogen demands for global food production are met by supplementing from nitrogen fertilizer obtained through the Haber-Bosch process, which is both polluting and expensive. Biological nitrogen fixation (BNF) requires the coordinated expression of ~20 accessory genes and the assembly of several complex metal cofactors which are irreversibly damaged by oxygen. We therefore have yet to succeed in synthetically engineering or heterologously expressing BNF in plants to boost crop-yields. The binding site of dinitrogen as well as the electronic structure and reactivity of the catalytic metal cluster of molybdenum-dependent nitrogenase are not fully understood. We have yet to determine the roles of several nif (nitrogen-fixation) genes involved in assembling nitrogenase, as well as several accesory genes associated with the genomic region. In addition to the most-studied molybdenum-dependent nitrogenase there are several alternative nitrogenases with unsual properties which are less well studied. My PhD will contribute to a better understanding of some of the less well-studied proteins associated with nitrogen fixation, particularly those involved in oxygen protection. Azotobacter is a model diazotroph with unusually high oxygen tolerance and amenability to genetic modification. In Azotobacter, molybdenum-dependent nitrogenase is conformationally protected from oxygen through the reversible binding of a protective ferredoxin (FeSII), forming a ternary complex which is oxygen-tolerant but inactive. In the Murray lab, we recently showed by crystallographic methods that the current hypothesis of the mechanism of FeSII binding involving a large confromational change is likely incorrect. The structure of the protective ternary complex has not been determined. On my PhD I will investigate the conformational protection of nitrogenase from oxygen by the structural charactisation of the nitrogenase:FeSII complex by single-particle electron microscopy. To this end I aim to isolate the labile native complex using partial purification by sucrose density gradients, co-immunoprecipitation or alternatively by affinity-purification of the his-tagged complex. I further aim to support our previous structural studies of the mechanism of FeSII binding by crystallising and structurally characterising several FeSII homologs with high sequence identity to FeSII, including those identified in Confluentimicrobium, Marinobacterium and Rhodobacter. I am also interested in purifying and characterising an unidentified protein of similar size to FeSII that is stably associated with the nitrogenase in Xanthobacter autotrophicus. Based on the unusually high oxygen tolerance of the nitrogenase this protein may might represent a new type of conformational protection and would be a fourth nitrogenase structural gene, which I aim to characterise using the above mentioned methods. I furthermore aim to study some of the less-well understood proteins involved in biosynthesis of metal clusters of molybdenum-dependent nitrogenase such as NifZ, NifT and NifW by heterologous expression, purification and structural characterisation. There are also several proteins associated with the nif region (or anf and vnf for the alternative nitrogenases) with unknown functions. For example, the oxidase Anf3 was recently characterised in this lab, while the roles of Anf1 and Anf2 remain to be determined and will contribute to our understanding of the iron-only nitrogenase.