Molecular mechanism of nitrogenase sequestration by a PII-protein couple

Abstract

The microbial process of N2-fixation is crucial for the planetary nitrogen cycle and biosphere, but requires substantial cellular energy resources. Here, we solved how energy-limited anaerobes regulate on-demand N2-fixation by sequestering their nitrogenase through the PII-nitrogen regulatory proteins NifI1 and NifI2. The nitrogenase was directly isolated from a methanogenic archaeon, with NifI proteins tightly bound. The crystal structure of the inhibited form, refined to 2.3-[A] resolution, reveals a supercomplex in which three NifI1,2 units made of a NifI1,2 heterohexamer captured three nitrogenases via tentacular T-loops. Additional structural information confirmed that NifI1,2 sits at the nitrogenase reductase-binding site, preventing N2-reduction. The presence of MgATP and 2-oxoglutarate releases NifI1,2 from the nitrogenase core via a conformational switch of the T-loops, provoking a steric repulsion and loss of contacts. The overall molecular depiction corroborates previous genetic, biochemical, and biophysical experiments, proposing that NifI1,2 disrupts the dynamic nitrogenase-reductase association, thereby interfering with electron delivery for N2-fixation and preventing ATP consumption. While ligand-binding mode and the T-loop conformational switch are expected to be conserved among NifI1,2-utilisers, the association mode with the nitrogenase comes in different flavours as a few substitutions in NifI2 break NifI1,2 intramolecular dimerisation in Methanosarcinales species, readjusting the supercomplex without altering the inhibition mechanism. With the NifI1,2 allosteric control dependent on the alarmone 2-oxoglutarate, anaerobes can effectively balance nitrogen-acquisition versus energy-expenditure, a regulatory switch that might be primitive and has been progressively lost in non-energy-limited aerobes, but could inspire biotechnological engineering to optimise ammonia bioproduction.