Heterologous Expression of Nitrogenase Iron Proteins and Investigation of its Extensive Potential

Nitrogenase is a key enzyme in the nitrogen cycle, and it is often produced by soil bacteria such as Azotobacter vinelandii, green sulfur bacteria, and cyanobacteria. Nitrogenase is a complex metalloenzyme responsible for converting nitrogen gas (N2) to bioavailable ammonia (NH3) under ambient conditions to ammonia, a transformation traditionally facilitated by industrial Haber-Bosch process under high temperature and pressure (≈450 °C, >200 atm). The iron protein of nitrogenase is one of two components and plays a key role as a reductase component to transfer electrons to the catalytic component of enzyme. Moreover, it has been discovered that iron protein can reduce carbon monoxide and carbon dioxide to short-chain hydrocarbons (alkanes and alkenes). There are two major research directions in the study of the nitrogenase system 1) understanding the structure and function of the enzyme as well as its complex metallocofactors to better understand N2 reduction and hydrocarbon formation and 2) the heterologous expression of the nitrogenase operon into different microorganisms for the development of microbe-based bioreactors and other biotechnology applications. This work focuses on the generation of a library of iron protein homologs that can be used as a toolbox for mechanistic investigations and to facilitate the heterologous expression of functional nitrogenase based systems. Moreover, this work explores the long-lasting debate of whether the Fe protein can utilize the [Fe4S4]0/2+ redox couple to support a two-electron transfer during substrate turnover which, therefore, is crucial for expanding our knowledge of the reaction mechanism of nitrogenase and the cellular energetics of nitrogenase-based processes