Assembly and Maturation of the Iron-Sulfur Clusters of Nitrogenases

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The nitrogenase system is a two-component enzyme machinery that catalyzes the reductive fixation of atmospheric dinitrogen as bioavailable ammonium. Its subcomplexes, the ATP-hydrolyzing Fe protein and the catalytic dinitrogenase, contain complex iron-sulfur clusters that require a dedicated, multi-step maturation machinery for their assembly. While the sequence of steps in nitrogenase cofactor maturation is largely understood, current knowledge about the molecular details and mechanisms of the individual factors remains scarce. With the ability to generate variants and deletion strains of the model diazotroph Azotobacter vinelandii, we will address these open question in the present proposal. We focus on the three major steps of nitrogenase cofactor maturation, the NifSU complex that builds a basic [4Fe:4S] fragment for further assembly, the radical/SAM enzyme NifB that rearranges two such clusters into a complex cofactor precursor, and the EN scaffold that completes maturation by inserting an apical heterometal, Mo or V. Affinity-tagged constructs for the three complexes are available of will be generated during the project, to be investigated by X-ray crystallography and spectroscopy, making use of the broad range of methods and tools within PP 1927. In addition, we will use spatially refined anomalous dispersion (SpReAD) refinement to gain experimental insights into several of the complex metal clusters that are investigated within the consortium.