Fe Protein-Independent Substrate Reduction by Nitrogenase MoFe Protein Variants

The reduction of substrates catalyzed by nitrogenase normally requires nucleotide-dependent Fe protein delivery of electrons to the MoFe protein, which contains the active site FeMo cofactor. Here, it is reported that independent substitution of three amino acids (beta-98(Tyr -> His), alpha-64(Tyr -> His), and beta-99(Phe -> His)) located between the P cluster and FeMo cofactor within the MoFe protein endows it with the ability to reduce protons to H-2, azide to ammonia, and hydrazine to ammonia without the need for Fe protein or ATP. Instead, electrons can be provided by the low-potential reductant polyaminocarboxylate-ligated Eu(II) (Em values of -1.1 to -0.84 V vs the normal hydrogen electrode). The crystal structure of the beta-98(Tr)?His variant MoFe protein was determined, revealing only small changes near the amino acid substitution that affect the solvent structure and the immediate vicinity between the P cluster and the FeMo cofactor, with no global conformational changes observed. Computational normal-mode analysis of the nitrogenase complex reveals coupling in the motions of the Fe protein and the region of the MoFe protein with these three amino acids, which suggests a possible mechanism for how Fe protein might communicate subtle changes deep within the MoFe protein that profoundly affect intramolecular electron transfer and substrate reduction.