A V‐Nitrogenase Variant Containing a Citrate‐Substituted Cofactor

Abstract Nitrogenases catalyze the ambient reduction of N 2 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 αβ 2 (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 N 2 reduction from H 2 evolution toward NH 3 formation and an opposite shift of CO reduction from hydrocarbon formation toward H 2 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 NH 3 /H 2 ratio upon citrate substitution suggests the possibility to modify the organic ligand for improved ammonia synthesis in the future.