A Comparative Analysis of the CO‐Reducing Activities of MoFe Proteins Containing Mo‐ and V‐Nitrogenase Cofactors

Abstract

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.

Reconstructing the evolutionary history of nitrogenases: Evidence for ancestral molybdenum‐cofactor utilization

“…Nitrogenase mediates the reduction of N 2 to NH 3 , a key step in nitrogen fixation (Hoffman et al, ; Seefeldt, Hoffman, & Dean, ). The metal dependence of nitrogenase, which impacts both catalytic properties (Eady, ; Harris, Yang, et al, ; Hu et al, ; Lee et al, ; Rebelein et al, ; Zheng et al, ) and ecological distribution (McRose, Zhang, Kraepiel, & Morel, ; Zhang et al, ), suggests a potential role for geochemical constraints on its evolution (Anbar & Knoll, ; Boyd, Hamilton, et al, ; Canfield et al, ; Raymond et al, ). Thus, understanding ancestral nitrogenase metal dependence can help resolve the early history of biological nitrogen fixation and, in a broader sense, the impact that ancient metal availabilities have had on the evolution of biologically essential metabolisms over Earth history (Anbar & Knoll, ; Moore, Jelen, Giovannelli, Raanan, & Falkowski, ).…”

“…Nevertheless, biochemical studies demonstrate variable catalytic properties among the three nitrogenase forms, including differential abilities to reduce alternative substrates (Harris, Lukoyanov, et al, ; Harris, Yang, Dean, Seefeldt, & Hoffman, ; Hu et al, ; Hu, Lee, & Ribbe, ; Zheng et al, ). These catalytic variations likely arise due to a combination of the aforementioned cofactor compositional differences and differences in the surrounding protein environment (Fixen et al, ; Harris, Yang, et al, ; Lee et al, ; Rebelein, Lee, Newcomb, Hu, & Ribbe, ; Zheng et al, ).…”

Assignment of protonated R-homocitrate in extracted FeMo-cofactor of nitrogenase via vibrational circular dichroism spectroscopy

“…Moreover, hydrocarbon formation like CH 4 , C 2 H 4 , C 2 H 6 by solvent-extracted cofactors proved CO can be reduced by cofactors in the presence of strong chemical reductants without the assistance of corresponding protein scaffolds 61,62 . But protein environment still is a major contributor in view of the activity of FeMo-co 63 . Namely, the catalytic activity of NMF-extracted FeMo-co can maintain in CO and N 2 reductions with the chelated mode of R-homocitrate, regarding the recent structures bound with CO and N 2 substrates 10,30 .…”

A V‐Nitrogenase Variant Containing a Citrate‐Substituted Cofactor

“…tween the wild-type and Vc luster-substitutedM oFe proteins (generated in vitro). [14] In both cases, the overall substrate-reducing activity and/or product profile were alteredu pon substitutiono ft he cofactor in the same protein scaffold,s uggesting af ine-tuning effect of the heterometal on the catalytic properties of the cofactor. Other than the heterometal, the organic compound of the cofactor-which is proposed to function in proton delivery during catalysis-was shown to have an impact on the substrate-reducing profileo fn itrogenase.…”