Luis Manuel Rubio Herrero y Carlos Echevarri Erasun
Date of presentation:
Faculty and University:
Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria y de Biosistemas. Universidad Politécnica de Madrid
Sobresaliente cum laude
Insights into nitrogenase biosynthesis obtained from thermophilic prokaryotes
Biological nitrogen fixation is an enzymatic process catalyzed by nitrogenase that reductively breaks the triple bond of nitrogen gas to render two ammonia molecules. 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. Mo-nitrogenase enzyme, as well as proteins required for its assembly and function, are encoded in 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 the Mo-nitrogenase. 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 loop constituted by residues C62 to E65 has a central role in the orchestration of SAM binding/cleavage and [Fe4S4] cluster stabilization.