The Mo- and V-nitrogenases are two homologous members of the nitrogenase family that are distinguished mainly by the presence of different heterometals (Mo or V) at their respective cofactor sites (M- or V-cluster). However, the V-nitrogenase is ~600-fold more active than its Mo counterpart in reducing CO to hydrocarbons at ambient conditions. Here, we expressed an M-cluster-containing, hybrid V-nitrogenase in Azotobacter vinelandii and compared it to its native, V-cluster-containing counterpart in order to assess the impact of protein scaffold and cofactor species on the differential reactivities of Mo- and V-nitrogenases toward CO. Housed in the VFe protein component of V-nitrogenase, the M-cluster displayed electron paramagnetic resonance (EPR) features similar to those of the V-cluster and demonstrated an ~100-fold increase in hydrocarbon formation activity from CO reduction, suggesting a significant impact of protein environment on the overall CO-reducing activity of nitrogenase. On the other hand, the M-cluster was still ~6-fold less active than the V-cluster in the same protein scaffold, and it retained its inability to form detectable amounts of methane from CO reduction, illustrating a fine-tuning effect of the cofactor properties on this nitrogenase-catalyzed reaction. Together, these results provided important insights into the two major determinants for the enzymatic activity of CO reduction while establishing a useful framework for further elucidation of the essential catalytic elements for the CO reactivity of nitrogenase.

The Mo and V nitrogenases are structurally homologous yet catalytically distinct in their abilities to reduce CO to hydrocarbons. Here we report a comparative analysis of the CO-reducing activities of the Mo- and V-nitrogenase cofactors (i.e., the M and V clusters) upon insertion of the respective cofactor into the same, cofactor-deficient MoFe protein scaffold. Our data reveal a combined contribution from the protein environment and cofactor properties to the reactivity of nitrogenase toward CO, thus laying a foundation for further mechanistic investigation of the enzymatic CO reduction, while suggesting the potential of targeting both the protein scaffold and the cofactor species for nitrogenase-based applications in the future.

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 VnfDGKCit, 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, VnfDGKCit 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.

The nitrogenase cofactors are structurally and functionally unique in biological chemistry. Despite a substantial amount of spectroscopic characterization of protein‐bound and isolated nitrogenase cofactors, electrochemical characterization of these cofactors and their related species is far from complete. Herein we present voltammetric studies of three isolated nitrogenase cofactor species: the iron–molybdenum cofactor (M‐cluster), iron–vanadium cofactor (V‐cluster), and a homologue to the iron–iron cofactor (L‐cluster). We observe two reductive events in the redox profiles of all three cofactors. Of the three, the V‐cluster is the most reducing. The reduction potentials of the isolated cofactors are significantly more negative than previously measured values within the molybdenum–iron and vanadium–iron proteins. The outcome of this study provides insight into the importance of the heterometal identity, the overall ligation of the cluster, and the impact of the protein scaffolds on the overall electronic structures of the cofactors.