Functional analysis of the Fe-nitrogenase components expressed in plant mitochondria

It has long been a goal for molecular biologists to engineer plants capable of performing their own nitrogen fixation, thereby reducing the reliance on industrially derived nitrogen fertilizer. Diazotrophic bacteria contain the nitrogenase enzyme, capable of converting atmospheric nitrogen (N2) to biologically available ammonia (NH3). Out of the three isoenzymes, Mo-, V-, and Fe-nitrogenase, the Fe-nitrogenase has a less complex biosynthetic and genetic componentry, only requiring the Fe ion at the enzymes active site, rather than the relatively scarce metals of molybdenum or vanadium. Nitrogenases are multi-component enzymes, containing oxygen-sensitive metalloclusters, that require access to ATP and electrons for function. Evidence is mounting that various components of nitrogenase are partially active when relocated to the inner matrix of mitochondria, where these exacting biochemical requirements may be provided. This thesis explores the co-expression, abundance, solubility, and interaction of a putative minimal pathway required for the Fe-nitrogenase, namely AnfD, AnfK, AnfH, AnfG, NifS, NifU, NifF, NifJ, NifB, and NifV when expressed in the mitochondrial matrix of the leaf cells of Nicotiana benthamiana. Import of the nitrogenase proteins into the mitochondrial matrix was confirmed via western blot, proteomic assessment, and the enrichment of intact plant mitochondria. Using affinity-based co-purification a protein-protein interaction between the mitochondrial targeted AnfD and AnfK proteins is demonstrated. A translational fusion between AnfD and AnfK, AnfD-linker-AnfK, improved the solubility and stoichiometric abundance of AnfD, presumably via a more stable protein-protein interaction in the plant mitochondria. Furthermore, the function of Fe-nitrogenase component AnfH isolated from mitochondria is demonstrated. Overall, results from this thesis establish that the Fe-nitrogenase is a promising candidate for engineering a functional nitrogenase into plant cells.