Insights into nitrogenase biosynthesis obtained from thermophilic prokaryotes

Abstract

Abstract Biological nitrogen fixation (BNF) is a high-energy cost enzymatic process that reductively breaks the triple bond of nitrogen gas (N2; N≡N) to render two ammonia (NH3) molecules. BNF is carried out, exclusively, by a few microorganisms belonging to the Bacteria and Archaea domains, known as diazotrophs. All diazotrophs fix N2 using a two-protein component nitrogenase enzyme. Based on its composition of metal cofactors, which are essential to activity, nitrogenases are classified as: molybdenum (Mo), vanadium (V) or iron-only (Fe) nitrogenases. All diazotrophs contain at least a Mo-nitrogenase, while some diazotrophs additionally contain V or Fe- nitrogenases. Therefore, the Mo-nitrogenase is the most abundant and is suggested to be the evolutionary predecessor of V- and Fe-nitrogenases. Mo-nitrogenase enzyme, as well as proteins required for its assembly, function, and regulation, are encoded in nitrogen fixation (nif) genes. The quantity and composition of a nif gene complement varies depending on the physiology and ecology of each diazotroph. However, a minimal set of six nif genes (nifHDKENB) has been established as essential criterium for being able to perform Mo-dependent N2 fixation. The present thesis investigates nitrogenase from the thermophilic bacterium Roseiflexus sp. RS-1, which appears depend only on nifHBDK. The NifH and NifDK nitrogenase structural components, and the biosynthetic protein NifB, have been characterized. Demonstration of in vitro activity present in purified Roseiflexus sp. nitrogenase components shows the existence of an alternative pathway for the biosynthesis of its active-site iron-molybdenum cofactor (FeMo-co). In Roseiflexus sp. FeMo-co biosynthesis seems to be NifEN-independent while NifDK has dual capability as FeMo-co maturase and nitrogenase enzyme. These results suggest that Roseiflexus sp. carries an enzyme complex likely resembling the predecessor of current Mo-nitrogenases before the events of duplication and divergence of nifDK and nifEN genes. Additionally, this thesis includes the first X-ray atomic structure resolution of a NifB protein. Because the Roseiflexus sp. NifB protein could not be crystallized, structure of the homologous NifB from Methanotrix thermoacetophila was solved in collaboration with the group of Dr. Yvain Nicolet at the Institute de Biologie Structurale. A novel [Fe4S4] cluster coordination involving two cysteine, one histidine and one glutamate residues was observed. NifB site directed mutagenesis and biochemical analyses performed as part of this thesis allowed us to refine a catalytic model for NifB-co synthesis, where a Lucía Payá Tormo loop constituted by residues C62 to E65 has a central role in the orchestration of SAM binding/cleavage and [4Fe-4S] cluster stabilization.