Zusammenfassung
The reduction by NADPH of the FAD and FMN redox centres in the isolated
flavin reductase domain of calmodulin-bound rat neuronal nitric
oxide synthase (nNOS) has been studied by anaerobic stopped-flow
spectroscopy using absorption and fluorescence detection. We show
by global analysis of time-dependent photodiode array spectra, single
wavelength absorption and NADPH fluorescence studies, that at least
four resolvable steps are observed in stopped-flow studies with
NADPH and that flavin reduction is reversible. The first reductive
step represents the rapid formation of an equilibrium between an
NADPH-enzyme charge-transfer species and two-electron-reduced enzyme
bound to NADP(+). The second and third steps represent further reduction
of the enzyme flavins and NADP(+) release. The fourth step is attributed
to the slow accumulation of an enzyme species that is inferred not
to be relevant catalytically in steady-state reactions. Stopped-flow
flavin fluorescence studies indicate the presence of slow kinetic
phases, the timescales of which correspond to the slow phase observed
in absorption and NADPH fluorescence transients. By analogy with
stopped-flow studies of cytochrome P450 reductase, we attribute
these slow fluorescence and absorption changes to enzyme disproportionation
and/or conformational change. Unlike for the functionally related
cytochrome P450 reductase, transfer of the first hydride equivalent
from NADPH to nNOS reductase does not generate the flavin di-semiquinoid
state. This indicates that internal electron transfer is relatively
slow and is probably gated by NADP(+) release. Release of calmodulin
from the nNOS reductase does not affect the kinetics of inter-flavin
electron transfer under stopped-flow conditions, although the observed
rate of formation of the equilibrium between the NADPH-oxidized
enzyme charge-transfer species and two-electron-reduced enzyme bound
to NADP(+) is modestly slower in calmodulin-depleted enzyme. Our
studies indicate the need for significant re-interpretation of published
kinetic data for electron transfer in the reductase domain of neuronal
nitric oxide synthase.
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