CO₂ Reduction Catalyzed by Nitrogenase: Pathways to Formate, Carbon Monoxide, and Methane

The reduction of N₂ to NH₃ by Mo-dependent nitrogenase at its active-site metal cluster FeMo-cofactor utilizes reductive elimination of Fe-bound hydrides with obligatory loss of H₂ to activate the enzyme for binding/reduction of N₂. Earlier work showed that wild-type nitrogenase and a nitrogenase with amino acid substitutions in the MoFe protein near FeMo-cofactor can catalytically reduce CO₂ by two or eight electrons/protons to carbon monoxide (CO) and methane (CH₄) at low rates. Here, it is demonstrated that nitrogenase preferentially reduces CO₂ by two electrons/protons to formate (HCOO^-) at rates >10 times higher than rates of CO₂ reduction to CO and CH₄. Quantum mechanical calculations on the doubly reduced FeMo-cofactor with a Fe-bound hydride and S-bound proton (E₂(2H) state) favor a direct reaction of CO₂ with the hydride ("direct hydride transfer" reaction pathway), with facile hydride transfer to CO₂ yielding formate. In contrast, a significant barrier is observed for reaction of Fe-bound CO₂ with the hydride ("associative" reaction pathway), which leads to CO and CH₄. Remarkably, in the direct hydride transfer pathway, the Fe-H behaves as a hydridic hydrogen, whereas in the associative pathway it acts as a protic hydrogen. MoFe proteins with amino acid substitutions near FeMo-cofactor (α-70^Val→Ala, α-195^His→Gln) are found to significantly alter the distribution of products between formate and CO/CH₄.