During catalysis the flavoprotein domain of neuronal nitric-oxide synthase (nNOS)
shuttles NADPH-derived reducing equivalents from FAD to FMN and then to the
P450-heme enabling heme-based oxygen activation and subsequent NO-synthesis. The
binding of Ca²⁺-activated calmodulin (Ca²⁺-CaM) to nNOS alleviates inhibition of
flavin mediated electron transfer within the diflavin domain, which is demonstrated by
the increase in the rate of 2,6-dichioroindoiphenol (DCIP) reduction by 2 to 3 fold and
that of cytochrome c³⁺ by 10 to 20 fold. To investigate the effect of the Ca²⁺-CaM on
the nNOS reductase activity, the steady-state kinetics of basal and CaM-stimulated
reduction of these two substrates was studied. Parallel initial velocity patterns
indicated that both substrates are reduced in a ping-pong mechanism. Product and
dead-end inhibition data with DCIP as the electron acceptor were consistent with a di
iso ping-pong bi-bi mechanism. In contrast, product and dead-end inhibition studies
with cytochrome c³⁺ as the second substrate were consistent with an iso (two-site) ping-pong
mechanism. Ca²⁺-CaM did not alter the proposed kinetic mechanisms; however,
it did effect to varying degrees the (k[subscript cat]/K[subscript]m) for the various substrates. The pH-dependence of basal and CaM-stimulated reduction of DCIP revealed that ionizable
groups involved in the binding of substrates and catalysis are not altered by Ca²⁺-CaM.
However, the activated cofactor does influence catalytic rate constants and/or ionizable
groups involved in cytochrome c³⁺ reduction. nNOS was found to abstract the pro-R
(A-side) hydrogen from NADPH. Primary deuterium isotope effects (NADP(D)) and
solvent isotope effects (SKIE) suggests that of the two half reactions, the reductive half
reaction involving NADPH oxidation limits the overall reaction rate, but that hydride
transfer to FAD is not the slow step. A small value of [supercript D](V/K)[subscript NADPH] (1.2-1.6) suggests hydride transfer is not the rate-limiting step within the reductive half-reaction. Large
solvent kinetic isotope effects (SKIE) were observed on (V/K)[subscript cytc] for basal and CaM stimulated
reduction of cytochrome c³⁺ suggesting that proton uptake from the solvent
limits the rate of the oxidative half-reaction. A small SKIE on V and (V/K)[subscript NADPH]
indicates that proton uptake does not limit the overall reaction rate. Proton inventory
analysis revealed multiple transition-state protons contributed to the observed SKIE.
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Previous issue date: 2001-04-24