Fig. 3: The NADPH-binding site and the SpNOX electron-transfer pathway.

a, Detailed view of the NADPH-binding site at the DH domain of SpNOX. Atoms within H-bond distance are marked with cyan dashed lines. FAD (gray) and amino acid side chains (coral) are shown as sticks. b, The lack of specificity toward NADPH in SpNOX can be explained by the absence of ionic interactions with the 2′-phosphate, unlike in eukaryotic NOXs including human DUOX1 (PDB 7D3F)⁹, in which R1424 and R1495 interact with the 2′-phosphate. c, The proposed electron-transfer path within SpNOX. Hemes, FAD, NADPH and the inter-heme hydrophobic residues are shown as sticks on the surface of SpNOX. Distances between the redox cofactors are represented as dashed black lines and were measured between the nicotinamide and the isoalloxazine ring (7.2 Å), between the isoalloxazine ring and the inner heme lower edge (9.9 Å) and between the edges of the inner and outer hemes (9.8 Å). d, F397 sits between the isoalloxazine ring of FAD and the nicotinamide ring of NADPH, impeding hydride transfer. e, The nicotinamide ring of NADPH moves closer to the isoalloxazine group of FAD in the F397A SpNOX mutant.