Structural basis of nitrogenase assembly and protection from oxygen

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.