Structural characterization of the P1+ intermediate state of the P-cluster of nitrogenase.

Nitrogenase is the enzyme that reduces atmospheric dinitrogen (N2) to ammonia (NH3) in biological systems. It catalyzes a series of single-electron transfers from the donor iron protein (Fe protein) to the molybdenum-iron protein (MoFe protein) that contains the iron-molybdenum cofactor (FeMo-co) sites where N2 is reduced to NH3 The P-cluster in the MoFe protein functions in nitrogenase catalysis as an intermediate electron carrier between the external electron donor, the Fe protein, and the FeMo-co sites of the MoFe protein. Previous work has revealed that the P-cluster undergoes redox-dependent structural changes and that the transition from the all-ferrous resting (PN) state to the two-electron oxidized P2+ state is accompanied by protein serine hydroxyl and backbone amide ligation to iron. In this work, the MoFe protein was poised at defined potentials with redox mediators in an electrochemical cell, and the three distinct structural states of the P-cluster (P2+, P1+, and PN) were characterized by X-ray crystallography and confirmed by computational analysis. These analyses revealed that the three oxidation states differ in coordination, implicating that the P1+ state retains the serine hydroxyl coordination but lacks the backbone amide coordination observed in the P2+ states. These results provide a complete picture of the redox-dependent ligand rearrangements of the three P-cluster redox states.

Structural characterization of the nitrogenase molybdenum-iron protein with the substrate acetylene trapped near the active site.

The biological reduction of dinitrogen (N2) to ammonia is catalyzed by the complex metalloenzyme nitrogenase. Structures of the nitrogenase component proteins, Iron (Fe) protein and Molybdenum­iron (MoFe) protein, and the stabilized complexes these component proteins, have been determined, providing a foundation for a number of fundamental aspects of the complicated catalytic mechanism. The reduction of dinitrogen to ammonia is a complex process that involves the binding of N2 followed by reduction with multiple electrons and protons. Electron transfer into nitrogenase is typically constrained to the unique electron donor, the Fe protein. These constraints have prevented structural characterization of the active site with bound substrate. Recently it has been realized that selected amino acid substitutions in the environment of the active site metal cluster (Iron­molybdenum cofactor, FeMo-co) allow substrates to persist even in the resting state. Reported here is a 1.70Å crystal structure of a nitrogenase MoFe protein α-96Arg➔Gln variant with the alternative substrate acetylene trapped in a channel in close proximity to FeMo-co. Complementary theoretical calculations support the validity of the acetylene interaction at this site and is also consistent with more favorable interactions in the variant MoFe protein compared to the native MoFe protein. This work represents the first structural evidence of a substrate trapped in the nitrogenase MoFe protein and is consistent with earlier assignments of proposed substrate pathways and substrate binding sites deduced from biochemical, spectroscopic, and theoretical studies.

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 (ß-98(Tyr→His), α-64(Tyr→His), and ß-99(Phe→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 ß-98(Tyr→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.