The Sixteenth Iron in the Nitrogenase MoFe Protein

Another iron in the fire: X-ray anomalous diffraction studies on the nitrogenase MoFe protein show the presence of a mononuclear iron site, designated as Fe16, which was previously identified as either Ca^(2+) or Mg^(2+). The position of the absorption edge indicates that this site is in the oxidation state +2. The high sequence conservation of the residues coordinated to Fe16 emphasizes the potential importance of the site in nitrogenase

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

Looking at Nitrogenase: Insights from Modern Structural Approaches

Nitrogenase, the primary biological source of fixed nitrogen, has been studied by various biochemical and biophysical methods to determine the mechanism of nitrogen reduction to ammonia. Previously, structural studies have contributed to determining the arrangement and identity of the unique metallocofactors of the as-isolated nitrogenase enzyme. Due to the multi-protein, dynamic nature of catalysis in nitrogenase, structurally capturing intermediates is not trivial. Recently, we have developed methods for preparing crystallographic samples of nitrogenase from active assay mixtures. The “out-of-assay” approach has yielded structures of small molecules bound to the active site cofactor, revealing an unexpected rearrangement of the belt sulfur atoms. The activity-based methods provide a framework for accessing non-resting states of the cofactor and introduce new questions surrounding the controlled binding and release of substrates. In the following, we discuss recent structural advances in the field and the novel directions for future activity-based research

Tracing the 'ninth sulfur' of the nitrogenase cofactor via a semi-synthetic approach.

The M-cluster is the [(homocitrate)MoFe7S9C] active site of nitrogenase that is derived from an 8Fe core assembled viacoupling and rearrangement of two [Fe4S4] clusters concomitant with the insertion of an interstitial carbon and a 'ninth sulfur'. Combining synthetic [Fe4S4] clusters with an assembly protein template, here we show that sulfite can give rise to the ninth sulfur that is incorporated in the catalytically important belt region of the cofactor after the radical S-adenosyl-L-methionine-dependent carbide insertion and the concurrent 8Fe-core rearrangement have already taken place. Based on the differential reactivity of the formed cluster species, we also propose a new [Fe8S8C] cluster intermediate, the L*-cluster, which is similar to the [Fe8S9C] L-cluster, but lacks the ninth sulfur from sulfite. This work provides a semi-synthetic tool for protein reconstitution that could be widely applicable for the functional analysis of other FeS systems