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 NO Synthase flavoprotein domains

摘要

NADPH-dependent dual-flavin enzymes provide electrons in many redox reactions, but what regulates their electron flux is 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 D.J. et al, 2009 FEBS J 276: 3959-3974). Here, we ran computer simulations of the kinetic model to determine if it could fit the experimentally-determined, pre-steady state and steady state traces of electron flux through the neuronal and endothelial NO synthase flavoproteins (nNOSr and eNOSr) to cytochrome c. We found the kinetic model accurately fit the experimental data. The simulations gave estimates for the ensemble rates of interflavin electron transfer and FMN domain conformational motion in nNOSr and eNOSr, provided the minimum rate boundary values, and predicted concentrations of the four enzyme species that cycle during catalysis. Our findings suggest that the rates of interflavin electron transfer and FMN domain conformational motion are counterbalanced so that both processes may limit electron flux through the enzymes. Such counterbalancing would allow a robust electron flux while keeping the rates of interflavin electron transfer and FMN domain conformational motion set at relatively slow levels.