Mechanical coupling in the nitrogenase complex

Abstract

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.

Structures of the nitrogenase complex prepared under catalytic turnover conditions

“…Originally, such half-reactivity was attributed to either partial inactivity of FeP molecules ( 19 ) or to the possible existence of an alternative interaction mode between FeP and MoFeP ( 20 ). Recent studies favored a model of negative cooperativity within a 2:1 FeP:MoFeP complex, whereby one of the bound FeP molecules suppresses ATP hydrolysis by the other bound FeP and the redox activity of the opposite αβ half of MoFeP ( 21 , 22 ). Our cryo-EM structures instead suggest that half-reactivity and negative cooperativity in nitrogenase arise from MoFeP binding to only one FeP molecule at a time during turnover.…”

Dissecting Electronic-Structural Transitions in the Nitrogenase MoFe Protein P-Cluster during Reduction

“…As established by kinetic and theoretical studies, correlated motions within the nitrogenase complex during turnover have an important function in enabling P-cluster mediated electron transfer to be integrated with catalysis. − , As shown here, during electron transfer, there are also discrete changes in P-cluster magnetic structure that are linked to changes in oxidation state. The EPR analysis and kinetic model are most consistent with these magnetic states originating from different P-cluster conformers during electron transfer and reduction of P 2+ to P + , which may function in the electron transfer mechanism within the nitrogenase complex during ammonia production …”

“… In the P + state, the S 1 sulfide is pentacoordinate and β-188Ser coordinates Fe 6 . The observation of structural changes in the MoFe protein P-cluster has been incorporated into conformational gating and mechanical coupling , electron transfer models. The model predicts that motions near the P-cluster and β-188Ser are coupled to “switch regions” in the Fe protein that steer structural interactions within the nitrogenase complex to enable electron delivery …”

An orphan protein drove the ecological expansion of nitrogen fixation

“…This residue is among the most well-connected within the G-D interaction network (Figure 3A-D), is critical for interface stability (Figure S7), and is among the most allosterically active, capable of generating long-distance effects that reach across opposite ends of the nitrogenase complex (Figure S11). These effects reflect the importance of the G-D interface in controlling the global motions of the nitrogenase complex and evoke similar, long-distance structural perturbations that are induced through the nitrogenase catalytic cycle 54,55 . The cleft itself provides an important anchor point for G-subunit binding and is present in both ancestral and representative extant nitrogenase structures (Figure 3A,B, Figure 4).…”