We seek to understand how nitrogenase and nitrogenase-like enzymes catalyze ATP-dependent multi-electron substrate reduction reactions. These enzymes share common structural ancestry and are made of homodimeric electron donor and heterotetrameric electron acceptor component proteins that transiently assemble in the presence of ATP to perform electron transfer. The heterotetrameric acceptor component possesses a2b2 symmetry. Thus, there are two identical halves and active sites arranged with mirrored symmetry. We uncovered that the two halves in nitrogenase and DPOR function in an asymmetric manner with catalytic events in one half allosterically controlling the other. Perturbing activities in one half stalls substrate reduction in the complex. To dissect the functional significance of this asymmetry and the mechanistic basis of allosteric control, we used CryoEM to capture snapshots of DPOR in the absence/presence of substrate, and during turnover in the presence of ATP.
The salient findings are: Substrate binding sets asymmetry in the protein complex. During turnover, only one donor component (BchL) binds to the tetrameric acceptor complex. We uncovered a new di-copper center in DPOR. The cluster is positioned between the two halves and essential for electron transfer. We uncovered the path of electron transfer and an extensive choreography of movements. Protection of the [4Fe-4S] cluster in the donor (BchL) by a disordered region dictates the engagement of the two proteins. Interestingly, in the absence of substrate, or when the di-Cu center is perturbed, asymmetry within DPOR is lost.
These findings enable us to postulate a role for the asymmetry where aromatic residues in one-half are aligned to promote electron transfer while misaligned in the other. ATP-hydrolysis within the electron donor component protein and the associated conformational changes drive alignment of the residues in the other half thereby priming it for activity. This architecture and regulation is likely conserved within all nitrogenase-like enzymes.