Abstract
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 (FeB) cofactor insertion into cytochrome c-dependent nitric oxide reductase (cNOR). cNOR catalyzes the NO reduction, a key step of bacterial denitrification. This work aimed at elucidating the specific mechanism of NorQD-catalyzed FeB insertion, and the general mechanism of the MoxR/VWA interacting protein families. 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. We present a tentative model for the mechanism of NorQD-catalyzed cNOR remodelling and suggest many of its features to be applicable to the whole MoxR/VWA family.
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- cNOR
- cytochrome c-dependent nitric oxide reductase
- MIDAS
- metal ion-dependent adhesion site
- VWA
- von Willebrand factor Type A
- qNOR
- quinol-dependent nitric oxide reductase
- HCuO
- heme-copper oxidase
- TM
- trans-membrane (helix)
- WT
- wildtype cNOR
- TMPD
- N,N,N′,N′-Tetramethyl-p-phenylene-diamine
- RBS
- ribosome binding site
- DDM
- n-Dodecyl β-D-maltoside
- cyt. c
- cytochrome c
- WB
- Walker B (motif).
