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. Designated NifDK^Cit , the catalytic component of this Mo-nitrogenase variant contains a citrate-substituted cofactor analogue. Activity analysis of NifDK^Cit 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 NifDK^Cit 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.