Insights into substrate binding at FeMo-cofactor in nitrogenase from the structure of an α-70Ile MoFe protein variant

The X-ray crystal structure is presented for a nitrogenase MoFe protein where the alpha subunit residue at position 70 (α-70Val) has been substituted by the amino acid isoleucine (α-70Ile). Substitution of α-70Val by α-70Ile results in a MoFe protein that is hampered in its ability to reduce a range of substrates including acetylene and N2, yet retains normal proton reduction activity. The 2.3 Å structure of the α-70Ile MoFe protein is compared to the α-70Val wild-type MoFe protein, revealing that the δ methyl group of α-70Val is positioned over Fe6 within the active site FeMo-cofactor. This work provides strong crystallographic support for the previously proposed model that substrates bind and are reduced at a single 4Fe–4S face of the FeMo-cofactor and that when α-70Val is substituted by α-70Ile access of substrates to Fe6 of this face is effectively blocked. Furthermore the detailed examination of the structure provides the basis for understanding the ability to trap and characterize hydrides in the variant, contributing significantly to our understanding of substrate access and substrate reduction at the FeMo-cofactor active site of nitrogenase.