| Nitrogenase is responsible for biological nitrogen fixation. The most studied nitrogenase is molybdenum nitrogenase. Combining genetic, biochemical, and spectroscopic methods, several advances toward understanding substrate binding and reduction mechanism of nitrogenase have been achieved. One major goal of this dissertation was aimed at understanding the N2 binding and reduction mechanism catalyzed by nitrogenase. A dihydride intermediate with two bridging hydrides bound to the FeMo-cofactor at E4 state was proposed as the key intermediate for N2 binding and activation. The 95Mo-ENDOR characterization of this E4 intermediate revealed that the Mo ion does not interact with the hydride ligands, suggesting that each hydride ligand bridges two Fe ions. When N2 binds to the FeMo-cofactor, one equivalent of H2 is produced. The N2-dependent incorporation of D2-derived deuterium into ethylene product of acetylene reduction during nitrogenase turnover strongly supports the proposed reductive elimination mechanism for N2 binding and activation at E4 state and confirms the mechanistic role of obligatory H2 loss. Once bound to the FeMo-cofactor, N2 was proposed to be stepwise reduced to ammonia through diazene and hydrazine intermediates. Two EPR-active common intermediates have been freeze-trapped during turnover of an altered MoFe protein with diazene, methyldiazene, and hydrazine as substrates. Pulsed ENDOR characterization of these two intermediates led to the assignment of the FeMo-cofactor bound ligands as -NH2 and NH3, respectively. These results support a proposed N2 reduction mechanism with unification of Lowe-Thorneley kinetic model and the alternating pathway. The other major goal was aimed at expanding nitrogenase catalysis toward other substrates. It was found that the alpha-70 amino acid residue of MoFe protein has a steric effect on the carbon monoxide (CO) coordination to the FeMo-cofactor. Further study revealed that CO can be reduced and coupled to form hydrocarbons by remodeled molybdenum nitrogenase. Moreover, carbon dioxide (CO2) was also found to be catalytically reduced to form methane by a remodeled molybdenum nitrogenase. The unprecedented formation of both propane and propylene was observed from the reductive coupling of CO2 and acetylene, supporting a plausible coupling mechanism based on two adjacent substrate binding sites. |
