Mechanical coupling in the nitrogenase complex

The enzyme nitrogenase reduces dinitrogen to ammonia utilizing electrons, protons, and energy obtained from the hydrolysis of ATP. Mo-dependent nitrogenase is a symmetric dimer, with each half comprising an ATP-dependent reductase, termed the Fe Protein, and a catalytic protein, known as the MoFe protein, which hosts the electron transfer P-cluster and the active-site metal cofactor (FeMo-co). A series of synchronized events for the electron transfer have been characterized experimentally, in which electron delivery is coupled to nucleotide hydrolysis and regulated by an intricate allosteric network. We report a graph theory analysis of the mechanical coupling in the nitrogenase complex as a key step to understanding the dynamics of allosteric regulation of nitrogen reduction. This analysis shows that regions near the active sites undergo large-scale, large-amplitude correlated motions that enable communications within each half and between the two halves of the complex. Computational predictions of mechanically regions were validated against an analysis of the solution phase dynamics of the nitrogenase complex via hydrogen-deuterium exchange. These regions include the P-loops and the switch regions in the Fe proteins, the loop containing the residue β-188Ser adjacent to the P-cluster in the MoFe protein, and the residues near the protein-protein interface. In particular, it is found that: (i) within each Fe protein, the switch regions I and II are coupled to the [4Fe-4S] cluster; (ii) within each half of the complex, the switch regions I and II are coupled to the loop containing β-188Ser; (iii) between the two halves of the complex, the regions near the nucleotide binding pockets of the two Fe proteins (in particular the P-loops, located over 130 Å apart) are also mechanically coupled. Notably, we found that residues next to the P-cluster (in particular the loop containing β-188Ser) are important for communication between the two halves.

Comment on “Structural evidence for a dynamic metallocofactor during N2 reduction by Mo-nitrogenase”

Kang et al. (Reports, 19 June 2020, p. 1381) report a structure of the nitrogenase MoFe protein that is interpreted to indicate binding of N2 or an N2-derived species to the active-site FeMo cofactor. Independent refinement of the structure and consideration of biochemical evidence do not support this claim.

An Unexpected P-Cluster like Intermediate En Route to the Nitrogenase FeMo-co

The nitrogenase MoFe protein contains two different FeS centers, the P-cluster and the iron-molybdenum cofactor (FeMo-co). The former is a [Fe8S7] center responsible for conveying electrons to the latter, a...

Synthesis of Nitrogenase by Paenibacillus Sabinae T27 in Presence of High Levels of Ammonia During Anaerobic Fermentation

BackgroundBiological nitrogen fixation catalyzed by nitrogenase is a high energy-intensive process, and thus nitrogenase synthesis and activity are inhibited by ammonium (NH4+). Microorganism fix nitrogen at high ammonium (30-300 mM) concentration has not been reported before.ResultsPaenibacillus sabinae T27, a Gram-positive, spore-forming diazotroph (N2-fixing microorganism, showed nitrogenase activities not only in low (0-4 mM) concentration of NH4+, but also in high (30-300 mM) concentration of NH4+, no matter whether the cells of this bacterium were grown in flask or in fermentor on scale cultivation. qRT-PCR and western blotting analysis supported that Fe protein and MoFe protein were synthesized under both low (0-4 mM) and high (30-300 mM) concentration of NH4+. Liquid chromatography-mass spectrometry（LC-MS）analysis revealed that MoFe protein purified form cultures grown in nitrogen-limited condition or nitrogen-excess condition was encoded by nifDK and Fe protein was encoded by both nifH and nifH2. The cross-reaction suggested the purified Fe and MoFe components from P. sabinae T27 grown in both nitrogen-limited and -excess conditions were active.ConclusionsOur results indicate that N2 fixation occurs in presence of high (30-300 mM) concentration of NH4+ in P. sabinae T27. Nitrogen fixation under both low and high concentration of NH4+ was catalyzed by the same nitrogenases and the Fe protein was encoded by both nifH and nifH2. Our study will provide a clue for studying the mechanisms on nitrogen fixation in presence of the high concentration of NH4+.

Using synthetic biology to overcome barriers to stable expression of nitrogenase in eukaryotic organelles

Engineering biological nitrogen fixation in eukaryotic cells by direct introduction ofnifgenes requires elegant synthetic biology approaches to ensure that components required for the biosynthesis of active nitrogenase are stable and expressed in the appropriate stoichiometry. Previously, the NifD subunits of nitrogenase MoFe protein fromAzotobacter vinelandiiandKlebsiella oxytocawere found to be unstable in yeast and plant mitochondria, respectively, presenting a bottleneck to the assembly of active MoFe protein in eukaryotic cells. In this study, we have delineated the region and subsequently a key residue, NifD-R98, fromK. oxytocathat confers susceptibility to protease-mediated degradation in mitochondria. The effect observed is pervasive, as R98 is conserved among all NifD proteins analyzed. NifD proteins from four representative diazotrophs, but not their R98 variants, were observed to be unstable in yeast mitochondria. Furthermore, by reconstituting mitochondrial-processing peptidases (MPPs) from yeast,Oryza sativa,Nicotiana tabacum, andArabidopsis thalianainEscherichia coli, we demonstrated that MPPs are responsible for cleavage of NifD. These results indicate a pervasive effect on the stability of NifD proteins in mitochondria resulting from cleavage by MPPs. NifD-R98 variants that retained high levels of nitrogenase activity were obtained, with the potential to stably target active MoFe protein to mitochondria. This reconstitution approach could help preevaluate the stability of Nif proteins for plant expression and paves the way for engineering active nitrogenase in plant organelles.

Structural evidence for a dynamic metallocofactor during N2 reduction by Mo-nitrogenase

The enzyme nitrogenase uses a suite of complex metallocofactors to reduce dinitrogen (N2) to ammonia. Mechanistic details of this reaction remain sparse. We report a 1.83-angstrom crystal structure of the nitrogenase molybdenum-iron (MoFe) protein captured under physiological N2 turnover conditions. This structure reveals asymmetric displacements of the cofactor belt sulfurs (S2B or S3A and S5A) with distinct dinitrogen species in the two αβ dimers of the protein. The sulfur-displaced sites are distinct in the ability of protein ligands to donate protons to the bound dinitrogen species, as well as the elongation of either the Mo–O5 (carboxyl) or Mo–O7 (hydroxyl) distance that switches the Mo-homocitrate ligation from bidentate to monodentate. These results highlight the dynamic nature of the cofactor during catalysis and provide evidence for participation of all belt-sulfur sites in this process.