Role for the nitrogenase MoFe protein alpha-subunit in FeMo-cofactor binding and catalysis

TWO components constitute Mo-dependent nitrogenase (EC 1.18.6.1)— the Fe protein (a homodimer encoded by nifH ) and the MoFe protein (an α 2 β 2 tetramer encoded by nifDK ). The MoFe protein provides the substrate-binding site 1–3 and probably coná-tains six prosthetic groups of two types—four Fe-S centres and two Fe- and Mo-containing cofactors 4,5 . To determine the distribution and catalytic function of these metalloclusters, we 6,7 and others 8 are attempting to change the catalytic and spectroscopic features of nitrogenase by substituting specific amino acids targeted as potential metallocluster ligands, particularly those to the FeMo-cofactor, which is responsible for the biologically unique electron paramagnetic resonance signal (S =3/2) of nitrogenase 9,10 , and is believed to be the N 2 -reducing site 11 . Here we describe mutant strains of Azotobacter vinelandii that have single a mi no-acid substitutions within the MoFe protein α-subunit. These substitutions alter both substrate-reduction properties and the unique electron paramagnetic resonance signal, indicating that the FeMo-cofactor is associated with both the α-subunit and the substrate-reducing site.