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

Nitrogenase converts N(2)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 anAzotobacter vinelandiistrain containing a deletion ofnifV, 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 H(2)evolution toward hydrocarbon formation and an opposite shift of N(2)reduction from NH(3)formation toward H(2)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-originatedS=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.

Automated summary

This study presents a biochemical and spectroscopic characterization of a Mo-nitrogenase variant with a citrate-substituted cofactor analogue, revealing the impact of citrate substitution on CO and N2 reduction pathways. The results suggest a crucial role of homocitrate in substrate reduction by nitrogenase and the potential to modulate product profiles of nitrogenase reactions through organic ligand substitution.