Abstract
Mo nitrogenase is the primary source of biologically fixed nitrogen, making this system highly interesting for developing new, energy efficient ways of ammonia production. Although heavily investigated, studies of the active site of this enzyme have generally been limited to spectroscopic methods that are compatible with the presence of water and relatively low protein concentrations. One method of overcoming this limitation is through lyophilization, which allows for measurements to be performed on solvent free, high concentration samples. This method also has the potential for allowing efficient protein storage and solvent exchange. To investigate the viability of this preparatory method with Mo nitrogenase, we employ a combination of electron paramagnetic resonance, Mo and Fe K-edge X-ray absorption spectroscopy, and acetylene reduction assays. Our results show that while some small distortions in the metallocofactors occur, oxidation and spin states are maintained through the lyophilization process and that reconstitution of either lyophilized protein component into buffer restores acetylene reducing activity.Graphic abstract
Highlights
Conversion of N 2 to NH3 is a critical biogeochemical process and essential for life on earth
We report the catalytic properties of the reconstituted forms of the two isolated protein components, MoFe and FeP, demonstrating the catalytic capacities of both proteins are retained through the lyophilization/reconstitution process
Assays were performed in triplicate over the course of 30 min Handling of protein samples for spectroscopic characterization is often challenging, potentially requiring buffer exchange and additional concentrating steps which invariably result in significant losses
Summary
Conversion of N 2 to NH3 is a critical biogeochemical process and essential for life on earth. In biology, this reaction is carried out exclusively by a small family of nitrogenase enzymes. The study of these enzymes has heavily focused on the Mo-dependent nitrogenase [11,12,13,14,15,16,17,18,19]
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