A V-Nitrogenase Variant Containing a Citrate-Substituted Cofactor

Nitrogenases catalyze the ambient reduction of N₂ and CO at its cofactor site. Herein we present a biochemical and spectroscopic characterization of an Azotobacter vinelandii V nitrogenase variant expressing a citrate-substituted cofactor. Designated VnfDGK^Cit, the catalytic component of this V nitrogenase variant has an αβ₂(δ) subunit composition and carries an 8Fe P* cluster and a citrate-substituted V cluster analogue in the αβ dimer, as well as a 4Fe cluster in the “orphaned” β-subunit. Interestingly, when normalized based on the amount of cofactor, VnfDGK^Cit shows a shift of N₂ reduction from H₂ evolution toward NH₃ formation and an opposite shift of CO reduction from hydrocarbon formation toward H₂ evolution. These observations point to a role of the organic ligand in proton delivery during catalysis and imply the use of different reaction sites/mechanisms by nitrogenase for different substrate reductions. Moreover, the increased NH₃/H₂ ratio upon citrate substitution suggests the possibility to modify the organic ligand for improved ammonia synthesis in the future.