The FAD-shielding residue Phe¹³⁹⁵ regulates neuronal nitric-oxide synthase catalysis by controlling NADP⁺ affinity and a conformational equilibrium within the flavoprotein domain

Phe¹³⁹⁵ stacks parallel to the FAD isoalloxazine ring in neuronal nitric-oxide synthase (nNOS) and is representative of conserved aromatic amino acids found in structurally related flavoproteins. This laboratory previously showed that Phe¹³⁹⁵ was required to obtain the electron transfer properties and calmodulin (CaM) response normally observed in wild-type nNOS. Here we characterized the F1395S mutant of the nNOS flavoprotein domain (nNOSr) regarding its physical properties, NADP⁺ binding characteristics, flavin reduction kinetics, steady-state and pre-steady-state cytochrome c reduction kinetics, and ability to shield its FMN cofactor in response to CaM or NADP(H) binding. F1395S nNOSr bound NADP⁺ with 65% more of the nicotinamide ring in a productive conformation with FAD for hydride transfer and had an 8-fold slower rate of NADP⁺ dissociation. CaM stimulated the rates of NADPH-dependent flavin reduction in wild-type nNOSr but not in the F1395S mutant, which had flavin reduction kinetics similar to those of CaM-free wild-type nNOSr. CaM-free F1395S nNOSr lacked repression of cytochrome c reductase activity that is typically observed in nNOSr. The combined results from pre-steady-state and EPR experiments revealed that this was associated with a lesser degree of FMN shielding in the NADP ⁺-bound state as compared with wild type. We conclude that Phe ¹³⁹⁵ regulates nNOSr catalysis in two ways. It facilitates NADP ⁺ release to prevent this step from being rate-limiting, and it enables NADP(H) to properly regulate a conformational equilibrium involving the FMN subdomain that controls reactivity of the FMN cofactor in electron transfer.