A kinetic model linking protein conformational motions, interflavin electron transfer and electron flux through a dual-flavin enzyme - simulating the reductase activity of the endothelial and neuronal nitric oxide synthase flavoprotein domains

NADPH-dependent dual-flavin enzymes provide electrons in many redox reactions, although the mechanism responsible for regulating their electron flux remains unclear. We recently proposed a four-state kinetic model that links the electron flux through a dual-flavin enzyme to its rates of interflavin electron transfer and FMN domain conformational motion [Stuehr DJ et al. (2009) FEBS J 276, 3959-3974]. In the present study, we ran computer simulations of the kinetic model to determine whether it could fit the experimentally-determined, pre-steady-state and steady-state traces of electron flux through the neuronal and endothelial NO synthase flavoproteins (reductase domains of neuronal nitric oxide synthase and endothelial nitric oxide synthase, respectively) to cytochrome c. We found that the kinetic model accurately fitted the experimental data. The simulations gave estimates for the ensemble rates of interflavin electron transfer and FMN domain conformational motion in the reductase domains of neuronal nitric oxide synthase and endothelial nitric oxide synthase, provided the minimum rate boundary values, and predicted the concentrations of the four enzyme species that cycle during catalysis. The findings of the present study suggest that the rates of interflavin electron transfer and FMN domain conformational motion are counterbalanced such that both processes may limit electron flux through the enzymes. Such counterbalancing would allow a robust electron flux at the same time as keeping the rates of interflavin electron transfer and FMN domain conformational motion set at relatively slow levels.