Crystallization of Nitrogenase Proteins

Nitrogenase is the only known enzymatic system capable of reducing atmospheric dinitrogen to ammonia. This unique reaction requires tightly choreographed interactions between the nitrogenase component proteins, the molybdenum–iron (MoFe)- and iron (Fe)-proteins, as well as regulation of electron transfer between multiple metal centers that are only found in these components. Several decades of research beginning in the 1950s yielded substantial information of how nitrogenase manages the task of N2 fixation. However, key mechanistic steps in this highly oxygen-sensitive and ATP-intensive reaction have only recently been identified at an atomic level. A critical part in any mechanistic elucidation is the necessity to connect spectroscopic and functional properties of the component proteins to the detailed three-dimensional structures. Structural information derived from X-ray diffraction (XRD) methods has provided detailed atomic insights into the enzyme system and, in particular, its active site FeMo-cofactor. The following chapter outlines the general protocols for the crystallization of Azotobacter vinelandii (Av) nitrogenase component proteins, with a special emphasis on different applications, such as high-resolution XRD, single-crystal spectroscopy, and the structural characterization of bound inhibitors

Homology modeling of nitrogenase iron proteins from three Frankia strains

The NifH protein contains an iron-sulfur cluster performing different functions during nitrogen fixation. Frankia is an actinomycete, entering into symbiotic association with a number of dicotyledonous plants and fixing nitrogen. The structure of the Frankia NifH protein was determined using homology modelling technique. Metal binding sites and functionally important regions of the protein were analyzed. Thiol ligands and active sites help in protein functioning and conformations. Structurally important nests were recognized. Clefts and cavities contain biologically important residues. Site-directed mutagenesis results reveal that mutations in functional residues hamper nitrogen fixation. The structure is rigid with an accessible surface for solvents. The structure is reliable offering insights into the 3D structural framework as well as structure-function relation of NifH protein