Journal
JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY
Volume 26, Issue 1, Pages 81-91Publisher
SPRINGER
DOI: 10.1007/s00775-020-01838-4
Keywords
Nitrogenase; X-ray absorption spectroscopy; Electron paramagnetic resonance; Lyophilization
Funding
- Max-Planck Society
- DFG [SPP 1927, DE 1877/1-1]
- Peter und Traudl Engelhorn Stiftung
- Projekt DEAL
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Mo nitrogenase is a key source of biologically fixed nitrogen, and investigations into its active site have been limited to spectroscopic methods. Lyophilization provides a method to overcome these limitations and allows for efficient protein storage and solvent exchange. Results show that the oxidation and spin states of metallocofactors are maintained through the lyophilization process, and reconstitution of the protein components restores activity.
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
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