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 (β-98Tyr→His, α-64Tyr→His, and β-99Phe→His) located between the P cluster and FeMo cofactor within the MoFe protein endows it with the ability to reduce protons to H2, 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 β-98Tyr→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.
Bibliographical notePublisher Copyright:
© 2015 American Chemical Society.