Dissecting the kinetics of the NADP+–FADH2 charge transfer complex and flavin semiquinones in neuronal nitric oxide synthase

• Full spectral scan (350–725 nm) for the NADPH-driven flavin reduction in nNOSrd.
• CT event (750 nm) and interflavin ET (600 nm) were monitored separately.
• Rate constants for charge transfer event were derived at 4 °C for the first time.

Electron flow within the neuronal nitric oxide synthase reductase domain (nNOSrd) includes hydride transfer from NADPH to FAD followed by two one-electron transfer reactions from FAD to FMN. We have used stopped flow spectrometry to closely monitor these electron transfer steps for both the wild type and the ΔG810 mutant of nNOSrd using a protocol involving both global analyses of the photodiode array spectral scans and curve fittings of single wavelength kinetic traces. The charge transfer complex and interflavin electron transfer events recorded at 750 nm and 600 nm, respectively, show the kinetics in different time frames. All electron transfer events are slow enough at 4 °C to enable measurements of rate constants even for the fast charge transfer event. To our knowledge this is the first time the rate constants for the charge transfer between NADP+ and FADH2 have been determined for NOS. These procedures allow us to conclude that (1) binding of the second NADPH is necessary to drive the full reduction of FMN and; (2) charge transfer and the subsequent interflavin electron transfer have distinct spectral features that can be monitored separately with stopped flow spectroscopy. These studies also enable us to conclude that interflavin electron transfer reported at 600 nm is not limiting in NOS catalysis.

Charge transfer between NADP+ and FADH2 is resolved from the interflavin electron transfer by stopped flow at low temperature.Figure optionsDownload as PowerPoint slide