Structural basis of nitrogenase assembly and protection from oxygen

氮酶催化二氮（N2）还原为氨（NH3）的动力学挑战。重氮营养微生物在氮循环中提供了大部分生物可利用氮。全球粮食生产对固定氮的需求不断增加，通过Haber-Bosch工艺获得的氮肥来补充，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，我们尚未成功地通过合成工程或在植物中异种表达BNF来提高作物产量。二氮的结合位点以及钼依赖氮酶的催化金属簇的电子结构和反应性尚不完全清楚。我们还没有确定几个nif（固氮）基因在组装氮酶中的作用，以及几个与基因组区域相关的辅助基因。除了研究最多的依赖钼的氮酶之外，还有几种具有不寻常性质的替代氮酶，但研究得较少。我的博士学位将有助于更好地理解一些研究较少的与固氮相关的蛋白质，特别是那些涉及氧气保护的蛋白质。固氮菌是一种典型的重氮营养菌，具有异常高的氧耐受性和对基因改造的适应性。在固氮细菌中，钼依赖的氮酶通过保护性铁氧还蛋白（FeSII）的可逆结合在构象上免受氧的影响，形成一个耐氧但无活性的三元配合物。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。保护性三元配合物的结构尚未确定。在我的博士学位上，我将通过单粒子电子显微镜对氮酶：FeSII复合物的结构表征来研究氮酶对氧的构象保护。为此，我的目标是分离不稳定的天然复合物，使用蔗糖密度梯度部分纯化，共免疫沉淀或亲和纯化his标记的复合物。为了进一步支持我们之前对FeSII结合机制的结构研究，我将对几个与FeSII具有高序列同一性的FeSII同源物进行结晶和结构表征，包括在confluentimicroium、Marinobacterium和Rhodobacter中发现的FeSII同源物。我还对纯化和表征一种与FeSII大小相似的未知蛋白质感兴趣，该蛋白质与自养黄杆菌（Xanthobacter autotrophicus）中的氮酶稳定相关。基于氮酶异常高的氧耐受性，该蛋白可能代表一种新型的构象保护，并且可能是第四个氮酶结构基因，我的目标是使用上述方法进行表征。此外，我的目标是通过异源表达、纯化和结构表征来研究一些不太为人所知的参与钼依赖氮酶金属簇生物合成的蛋白质，如NifZ、NifT和NifW。还有一些与nif区（或替代氮酶的anf和vnf）相关的功能未知的蛋白质。例如，氧化酶Anf3最近在这个实验室中被表征，而Anf1和Anf2的作用仍有待确定，这将有助于我们对纯铁氮酶的理解。

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.