FIGURE 8.

Schematic illustration of NADPH binding to and NADP⁺ release from CYPOR. For clarity, only the FAD/NADP(H)-binding domain of CYPOR is shown. NADPH enters oxidized CYPOR (state 1, modeled after the structure of 147CC514, which lacks bound NADP(H)). NADPH initially binds to the enzyme in a folded conformation, such that the adenine ring and the nicotinamide ring almost stack on each other. Upon binding of NADPH, the Asp⁶³² loop of CYPOR moves away to allow NADPH to bind (state 2, modeled from the structure of 147C/C514-NADP⁺). Once the NADPH binds, the Asp⁶³² loop moves in and displaces the nicotinamide ring, resulting in the ribityl-nicotinamide moiety extending to search for the proper binding site for hydride transfer, while keeping the ADP-PP_i anchor at the arginine site. The indole ring of Trp⁶⁷⁷ rotates to be ready to move away from the flavin ring (state 3, the structure of wild type; PDB code 1JA1). The indole ring moves away to make room for the nicotinamide ring to bind at the re-side of the isoalloxazine ring (state 4, modeled after the structure of the mutant W677X, in which the two penultimate residues of CYPOR, Trp⁶⁷⁷ and Ser⁶⁷⁸, were truncated, PDB code 1JA0). Hydride transfer occurs; FAD is reduced, and NADPH becomes NADP⁺ (state 5, from PDB code 1JA0). Once the flavin is reduced and the nicotinamide is oxidized, the nicotinamide ring moves out and the indole ring returns to the re-face of the FAD ring (state 6, PDB code 1JA1). As the Asp⁶³² loop moves away allowing for the nicotinamide ring to fold back, NADP⁺ adopts the folded conformation poised to dissociate from the enzyme (state 7, the structure of 147C/C514-NADP⁺). The Asp⁶³² loop moves back closer to where the NADP⁺ pyrophosphate-2′-AMP lies, causing steric hindrance as well as electrostatic repulsion, resulting in dissociation of the cofactor from CYPOR. The enzyme now returns to state 1 and the cycle repeats.