Neuronal Nitric Oxide Synthase: Substrate and Solvent Kinetic Isotope Effects on the Steady-State Kinetic Parameters for the Reduction of 2,6-Dichloroindophenol and Cytochrome c3+

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Abstract
The neuronal nitric oxide synthase (nNOS) basal and calmodulin- (CaM-) stimulated reduction of 2,6-dichloroindophenol (DCIP) and cytochrome c3+ follow ping-pong mechanisms [Wolthers and Schimerlik (2001) Biochemistry40, 4722−4737]. Primary deuterium [NADPH(D)] and solvent deuterium isotope effects on the kinetic parameters were studied to determine rate-limiting step(s) in the kinetic mechanisms for the two substrates. nNOS was found to abstract the pro-R (A-side) hydrogen from NADPH. Values for DV and D(V/K)NADPH were similar for the basal (1.3−1.7) and CaM-stimulated (1.5−2.1) reduction of DCIP, while DV (2.1−2.8) was higher than D(V/K)NADPH (1.1−1.5) for cytochrome c3+ reduction with and without CaM. This suggests that the rate of the reductive half-reaction (NADPH oxidation) rather than that of the oxidative half-reaction (reduction of DCIP or cytochrome c3+) limits the overall reaction rate. A value for D(V/K)NADPH close to 1 indicates the intrinsic isotope effect on hydride transfer is suppressed by a slower step in the reductive half-reaction. The oxidative half-reaction is insensitive to NADPD isotope effects as both D(V/K)DCIP and D(V/K)cytc equal 1 within experimental error. Large solvent kinetic isotope effects (SKIE) observed for (V/K)cytc for basal (∼8) and CaM-stimulated (∼31) reduction of cytochrome c3+ suggest that proton uptake from the solvent limits the rate of the oxidative half-reaction. This step does not severely limit the overall reaction rate as D2OV equaled 2 and D2O(V/K)NADPH was between 0.9 and 1.3 for basal and CaM-stimulated cytochrome c3+ reduction.

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