UC San Diego

Nitrogen is an essential component of many biomolecules such as DNA and proteins. Biological nitrogen fixation is carried out by the enzyme nitrogenase. This multi-electron reduction requires the precise timing of electron and proton flow from the reductase, Fe-protein, through the [8Fe-7S] P-cluster to the catalytic site, FeMo-cofactor, located in the catalytic protein, MoFe-protein. Electron flow through nitrogenase is a dynamic process controlled by a conformational “gate” in which structural changes in MoFe-protein promote the accumulation of electrons at the FeMo-cofactor. Elucidating the mechanism thus requires a detailed understanding of structural changes that occur during catalysis. I have investigated the dynamics involved in nitrogenase catalysis, focusing on (1) the P-cluster’s dynamic role in orchestrating electron transfer to FeMo-cofactor and (2) structural characterization of the nitrogenase complex during N2 reduction. The experimental results discussed in Chapters 2 and 3 of this dissertation describe how the P-cluster has evolved to rest on the brink of stability, and that mutations to the redox-switchable, O-based P-cluster ligand (serine or tyrosine) result in a cluster that can reversibly lose Fe atoms. Furthermore, the O-based ligand protects the P-cluster from oxidative stress in vivo, is required for efficient diazotrophic growth under Fe-limiting conditions, and protects the P-cluster from metal exchange in vitro. These results indicate the native flexibility of the P-cluster are vital to its function in timing electron transfer to the active site. In Chapter 4, I discuss structural characterization of the nitrogenase complex at high-resolution during catalytic N2 reduction with cryoEM. Many structures were determined: structures of the complex during turnover at 2.6Å and 2.7Å, free MoFeP at 1.9Å, and ADP.AlF4—-inhibited complexes at 2.4Å and 2.8Å. Taken together, these structures have provided valuable insights into the mechanism of biological nitrogen fixation. The complex formed during turnover has a 1:1 Fe-protein:MoFe-protein stoichiometry, implying that there is negative cooperativity between the two halves of the complex. Furthermore, conformational differences between the two halves of MoFe-protein reveal previously unobserved conformational changes, which may be a part of the conformational “gate”. These observations provide critical insights into the dynamics required for nitrogenase function.