Characterization of a Mo‐Nitrogenase Variant Containing a Citrate‐Substituted Cofactor

Nitrogenase converts N2 to NH3, and CO to hydrocarbons, at its cofactor site. Herein, we report a biochemical and spectroscopic characterization of a Mo‐nitrogenase variant expressed in an Azotobacter vinelandii strain containing a deletion of nifV, the gene encoding the homocitrate synthase. Design...

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Published in:Chembiochem : a European journal of chemical biology 2021-01, Vol.22 (1), p.151-155
Main Authors: Liedtke, Jasper, Lee, Chi Chung, Tanifuji, Kazuki, Jasniewski, Andrew J., Ribbe, Markus W., Hu, Yilin
Format: Article
Language:English
Subjects:
Ammonia
Azotobacter
Carbon monoxide
Citric acid
cofactors
Gene deletion
Homocitrate synthase
Hydrocarbons
Hydrogen evolution
molybdenum nitrogenase
Nitrogenase
Reduction
Substitutes
Substrates
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Summary:Nitrogenase converts N2 to NH3, and CO to hydrocarbons, at its cofactor site. Herein, we report a biochemical and spectroscopic characterization of a Mo‐nitrogenase variant expressed in an Azotobacter vinelandii strain containing a deletion of nifV, the gene encoding the homocitrate synthase. Designated NifDKCit, the catalytic component of this Mo‐nitrogenase variant contains a citrate‐substituted cofactor analogue. Activity analysis of NifDKCit reveals a shift of CO reduction from H2 evolution toward hydrocarbon formation and an opposite shift of N2 reduction from NH3 formation toward H2 evolution. Consistent with a shift in the Mo K‐edge energy of NifDKCit relative to that of its wild‐type counterpart, EPR analysis demonstrates a broadening of the line‐shape and a decrease in the intensity of the cofactor‐originated S=3/2 signal, suggesting a change in the spin properties of the cofactor upon citrate substitution. These observations point to a crucial role of homocitrate in substrate reduction by nitrogenase and the possibility to tune product profiles of nitrogenase reactions via organic ligand substitution. Substituting citrate for homocitrate in the M‐cluster of Mo‐nitrogenase shifts CO reduction from H2 evolution toward hydrocarbon formation and conversely N2 reduction from NH3 formation toward H2 evolution. This suggests a crucial role for homocitrate in substrate reduction by nitrogenase and the possibility to modulate product profiles of nitrogenase reactions by substituting the organic ligand of the cofactor.
ISSN:1439-4227
1439-7633
DOI:10.1002/cbic.202000598