Investigation of Nitrogenase Variants
- Author(s): Newcomb, Megan
- Advisor(s): Ribbe, Markus
- et al.
Nitrogenases are complex metalloenzymes that catalyze the reduction of nitrogen gas (N2) to form bioavailable ammonia (NH3) under ambient conditions. These enzymes can also reduce carbon monoxide (CO), a potent pollutant gas, into small hydrocarbon products. The molybdenum (Mo) and vanadium (V) nitrogenases are two homologous members of the nitrogenase family that are both comprised of a multi-subunit protein scaffold and a complex active site metallocofactor made up of an iron-sulfur core and an organic ligand.
The Mo- and V-nitrogenases demonstrate different capabilities with respect to the reduction of N2 and CO. Specifically, the Mo-nitrogenase is about twice as active as V-nitrogenase at producing NH3 from N2. Interestingly, the V-nitrogenase is ~600-fold more active than the Mo-nitrogenase in reducing CO to hydrocarbon products. These reactivity differences likely stem from dissimilarities in the protein scaffolds and active site cofactor properties of the Mo- and V-nitrogenase variants, and the work described in this dissertation probes the roles of these aspects through biochemical and spectroscopic characterization. A hybrid protein made up of the V-nitrogenase protein scaffold and the Mo-nitrogenase cofactor was created in vivo through the manipulation of bacterial growth conditions. The substrate reduction activity of the purified hybrid protein indicates that the protein scaffold of V-nitrogenase is the primary contributor to the observed differences in its CO-reducing capabilities. Additional work was conducted with a mutant of V-nitrogenase that produced an active site cofactor with an alternative organic ligand. The modified V-nitrogenase and its isolated cofactor demonstrated an increased production of NH3 from N2, which indicates that the organic ligand plays an important role in protonation of various small molecule substrates. The results of this work highlight the importance of the V-nitrogenase protein scaffold and its organic ligand in the production of hydrocarbon products from CO and NH3 from N2.