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

Abstract

The X-ray crystal structure is presented for a nitrogenase MoFe protein where the alpha subunit residue at position 70 (alpha-70(Val)) has been substituted by the amino acid isoleucine (alpha-70(Ile)). Substitution of alpha-70(Val) by alpha-70(Ile) results in a MoFe protein that is hampered in its ability to reduce a range of substrates including acetylene and N(2), yet retains normal proton reduction activity. The 2.3A structure of the alpha-70(Ile) MoFe protein is compared to the alpha-70(Val) wild-type MoFe protein, revealing that the delta methyl group of alpha-70(Val) 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 alpha-70(Val) is substituted by alpha-70(Ile) 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.

Figure 1:

Stereoviews of the protein environment surrounding the FeMo-cofactor. Shown is a portion of α-carbon trace and select amino acid side chains for both the α-70^Ile (magenta) and wild type (cyan) MoFe proteins in two orientations (A and B) separated by an ~90? rotation. The protein backbone is shown as ribbons, with the residues immediately surrounding FeMo-cofactor from the α-subunit and the FeMo-cofactor shown in line angle representations with the exception of either the α-70^Val (wild-type) side chain and the α-70^Ile (variant) side chain shown in space filling / van der Waals representations. Fe atoms are colored in rust, S in yellow, Mo in black,and X in cyan.

Figure 2:

Stereoview of the 2Fo-Fc electron density maps (1.0σ) of the region surrounding the FeMo-cofactor and the adjacent α-70^Ile residue. The protein backbone is shown in ribbons with residues in the FeMo-cofactor environment from the α-subunit are shown in line angle representations color coded with Fe in rust, S in yellow, C in grey, N in blue, O in red, and Mo in black.

Figure 3:

FeMo-cofactor binding site in the the α-70^Ile MoFe protein structure showing the location of the surrounding water molecules represented as red spheres. The residues in the FeMo-cofactor and surrounding protein environment shown with line angle representations color coded as in Figure 2..