Caracterización de una nueva proteína hipertermófila NifB y su papel en el mecanismo de síntesis de NifB-co, precursor del grupo metálico de FeMo-co

摘要

Nitrogen is a limiting nutrient for plant growth, both in natural and agricultural ecosystems. Biological nitrogen fixation (BNF) is a process catalyzed by metallo enzymes called nitrogenases. There are three types of nitrogenases depending on the metal group present at the active site, the most abundant being the Mo-nitrogenase, which carries the iron-molybdenum cofactor (FeMo-co). The genes encoding the nitrogenase related proteins are found exclusively in prokaryotes and are organized into operons, termed nif. Mo-nitrogenase is a two-component system composed of a NifD2K2 heterodimer, where each NifDK half carries two metal cofactors (one Pcluster and one FeMo-co), and a NifH homodimer that carries a [Fe4S4] at the subunit interface. FeMo-co is arguably the most complex modified [Fe-S] cluster in Biology. Its biosynthesis requires highly specific reactions that are tightly regulated. NifB catalyzes the first essential reaction in FeMo-co biosynthesis. Interestingly, NifB activity is required for all nitrogenases. NifB is a radical S-adenosylmethionine (SAM) enzyme that synthesizes the [Fe8S9C] precursor of FeMo-co, named NifB-co. Carrying a signature CxxxCxxC motif at its N terminus, which houses the SAMbinding [Fe4S4] cluster (designated as SAM cluster), NifB also contains a number of additional ligands that could co-ordinate the entire complement of iron (Fe) atoms of NifB-co. Due to its extreme sensitivity to O2 and inherent protein instability, NifB proteins must be purified under strict anaerobic conditions by using affinity chromatography methods. Our studies and overlapping studies of other group show that, in the presence of SAM, NifB is capable of inserting a carbide atom concomitant with the coupling of two [Fe4S4] clusters to form a [Fe8S9C] cluster, which represents the all-iron core of the FeMo-co. In this thesis, we have contributed and developed methods for NifB purification from cells of the facultative anaerobic non-nitrogen fixing bacterium Escherichia coli recombinantly expressing a nifB gene of thermophilic origin (NifBMi). The stability of NifBMi allowed a deep biochemical and spectroscopic characterization of this enzyme. In our study, we demonstrated that NifBMi is a radical SAM enzyme able to reductively cleave SAM to 5′- deoxyadenosine radical and is competent in FeMo-co maturation. Furthermore we characterize the metal cluster organization of this protein through electron paramagnetic resonance spectroscopy and electron spin echo envelope modulation, offering mechanistic insights into NifB-co synthesis.