Identification of an Fe protein residue (Glu(146)) of Azotobacter vinelandii nitrogenase that is specifically involved in FeMo cofactor insertion

The Fe protein of nitrogenase has three separate functions. Much is known a bout the regions of the protein that are critical to its function as an ele ctron donor to the MoFe protein, but almost nothing is known about the regi ons of the protein that are critical to its functions in either FeMo cofact or biosynthesis or FeMo cofactor insertion. Using computer modeling and inf ormation obtained from Fe protein mutants that were made decades ago by che mical mutagenesis, we targeted a surface residue GlU(146) as potentially be ing involved in FeMo cofactor biosynthesis and/or insertion, The Azotobacte r vinelandii strain expressing an E146D Fe protein variant grows at similar to 50% of the wild type rate. The purified E146D Fe protein is fully funct ional as an electron donor to the MoFe protein, but the MoFe protein synthe sized by that strain is partially (similar to 56%) FeMo cofactor-deficient, The E146D Fe protein is fully functional in an in vitro FeMo cofactor bios ynthesis assay, and the strain expressing this protein accumulates "free" F eMo cofactor. Assays that compared the ability of wild type and E146D Fe pr oteins to participate in FeMo cofactor insertion demonstrate, however, that the mutant is severely altered in this last reaction. This is the first kn own mutation that only influences the insertion reaction.