UC San Diego

The Haber-Bosch process carries an enormous industrial and agricultural importance but also has a largely negative economic and environmental impact. Understanding the mechanistic details of biological nitrogen fixation catalyzed by the enzyme nitrogenase would be beneficial both for designing cleaner or more efficient catalysts for small molecule activation and for understanding biological multi-electron/proton redox processes. The enzyme's catalytic mechanism, however, is poorly understood because it relies upon ATP hydrolysis and complex protein-protein interactions to coordinate electron transfer and substrate activation. We have proposed that uncoupling MoFeP catalysis from ATP- and FeP-dependent electron transfer, specifically through light-driven electron injection, would enable the direct study of catalysis at the enzyme active site and the population of discrete reaction intermediates for structural or spectroscopic investigation. The experimental results discussed herein describe the design and characterization of a photosensitized MoFeP capable of reducing substrates independent of ATP hydrolysis and FeP, which have previously been believed essential for catalytic turnover. To achieve light-driven electron injection and substrate activation, MoFeP variants were labeled with a Ru photosensitizer at three different exposed cysteine residues on the protein surface. A particular MoFeP-Ru construct with the Ru photosensitizer located directly above the P-cluster was found to catalyze the 2-electron reduction of protons and acetylene in a light-dependent manner. A modified version of the same construct was able to catalyze the 6-electron reduction of HCN into CH₄ and likely also NH₃. Currently, none of the MoFeP-Ru constructs can catalyze the light-driven reduction of N₂ to NH₃, most likely as a result of inefficient electron transfer to the FeMoco in the absence of FeP. In light of recent findings that certain amino-acid substitutions in MoFeP may enable efficient electron transfer into the MoFeP active site (FeMoco) in the absence of FeP, we have initiated efforts focused on modifying the MoFeP through site-directed mutagenesis. Incorporating these substitutions into our MoFeP-Ru constructs should increase the efficiency of electron transfer to FeMoco such that light-driven N₂ reduction is realized and discrete catalytic intermediates for this reaction can be populated in sufficient quantities for structural or spectroscopic examination