UC Irvine

The iron (Fe) protein is one of two components involved in the biological fixation of dinitrogen (N2) to bioavailable ammonia (NH3) by nitrogenase. The physiological roles of the Fe protein include transferring electrons to the catalytic component and assisting in the bioassembly of the two metalloclusters found within the catalytic component. The Fe protein has also been shown to adventitiously reduce CO2. The goal of this work has been to heterologously express and characterize Fe protein homologs according to their physiological and adventitious functions. This dissertation is divided into two sections, comprising multiple chapters, based on these functions.

Section 1 begins by comparing the molybdenum (Mo-) and vanadium (V-) nitrogenase Fe proteins from Methanosarcina acetivorans (MaNifH and MaVnfH, respectively) to the Fe proteins from the model organism Azotobacter vinelandii (AvNifH and AvVnfH, respectively). Characterization includes comparing the size of the proteins, the behavior of the [Fe4S4] cluster of the protein, and the ability of the homologs to assist in the bioassembly of the catalytic component’s metalloclusters. Next, there is a discussion about a hybrid nitrogenase system, created by pairing MaVnfH with the catalytic component of A. vinelandii, which resulted in the ability to trap the alternate V-nitrogenase substrate, CO, on the catalytic cofactor. Also included are a brief chapter that reports the MgADP-bound crystal structure of MaVnfH and a miscellaneous chapter, which contains preliminary results regarding the characterization of five additional Mo-nitrogenase Fe protein homologs.

The first chapter of Section 2 reports that MaNifH is able to reduce CO2 beyond CO and also generate hydrocarbons, which is further than the A. vinelandii counterparts. Consequently, a CO reduction assay was created, and this section focuses on the ability of Fe protein homologs to reduce both CO2 and CO. It was determined that CO2 and CO reduction is an intrinsic property of [Fe4S4] clusters. The final chapter compares the product profiles of different Fe protein homologs and compares the crystal structures of these Fe proteins to gain insights into what may cause these differences.