Background NorQ, a member of the MoxR-class of AAA+ ATPases, and NorD, a protein containing a Von Willebrand Factor Type A (VWA) domain, are essential for non-heme iron (Fe_B) cofactor insertion into cytochrome c-dependent nitric oxide reductase (cNOR). cNOR catalyzes NO reduction, a key step of bacterial denitrification. This work aimed at elucidating the specific mechanism of NorQD-catalyzed Fe_B insertion, and the general mechanism of the MoxR/VWA interacting protein families.

Results We show that NorQ-catalyzed ATP hydrolysis, an intact VWA domain in NorD, and specific surface carboxylates on cNOR are all features required for cNOR activation. Supported by BN-PAGE, low-resolution cryo-EM structures of NorQ and the NorQD complex show that NorQ forms a circular hexamer with a monomer of NorD binding both to the side and to the central pore of the NorQ ring. Guided by AlphaFold predictions, we assign the density that “plugs” the NorQ ring pore to the VWA domain of NorD with a protruding “finger” inserting through the pore and suggest this binding mode to be general for MoxR/VWA couples.

Conclusions Based on our results, we present a tentative model for the mechanism of NorQD-catalyzed cNOR remodeling and suggest many of its features to be applicable to the whole MoxR/VWA family.

ATPases associated with diverse cellular activities (AAA+-ATPases) catalyze a wide range of remodeling events in all phyla. MoxR AAA+-ATPases co-operate with VWA (von Willebrand factor type A) domain containing proteins, often to remodel their targets enabling insertion of metal ions. The mechanism of action of the MoxR-VWA complexes are poorly understood. We have solved cryo-EM structures of the MoxR AAA+-ATPase NorQ in complex with its VWA domain partner protein NorD. NorQ and NorD interact primarily via a) a finger protruding from the VWA domain, inserting into the central pore of the NorQ hexamer and b) the C- terminus of NorD interacts with a post sensor 1 loop in NorQ. Based on structural and biochemical data, we propose a mechanism where the NorQD complex ‘twists’ and stretches the NorD linker to enable metal insertion into its target nitric oxide reductase (NOR). 

The heme-copper oxidase nitric oxide reductase catalyzes the reduction of nitric oxide into nitrous oxide in prokaryotic denitrification chains. The cNOR catalytic site consists of one heme b₃ and one Fe_B, both required for catalytic activity. Fe_B insertion is dependent on the two chaperones NorQ, a MoxR AAA+ ATPase and NorD, a VWA domain protein. Here, we investigate the interactions between the NorQD chaperones and their cNOR target, using co-overexpression experiments, site-directed mutagenesis and cryo-EM. In addition, we show that the two cNOR subunits, NorC and NorB, can be expressed and purified separately, and that chaperone mediated Fe_B insertion is dependent on both subunits. 

Cytochrome c-dependent nitric oxide reductase (cNOR) catalyzes the reduction of NO into nitrous oxide (N₂O), a strong greenhouse gas released from denitrifying microorganisms. The cNOR active site holds an essential non-heme iron, Fe_B, inserted using the chaperone complex NorQD. However, in Thermus thermophilus, the cNOR (TtcNOR) cluster lacks the norQD genes. Here we investigated Fe_B insertion into TtcNOR and characterized and compared TtcNOR expressed in Escherichia coli to that natively produced. We show that Fe_B is present in the natively produced TtcNOR only. Analysis of cNOR operon sequences suggests that a hydrophilic K-pathway analogue is present in cNORs that do not rely on NorQD for iron insertion. We discuss the implications of our data for the evolution of the NOR family.