Deletion of the autoregulatory insert modulates intraprotein electron transfer in rat neuronal nitric oxide synthase

Abstract 

Comparative CO photolysis kinetics studies on wild-type and autoregulatory (AR) insert-deletion mutant of rat nNOS holoenzyme were conducted to directly investigate the role of the unique AR insert in the catalytically significant FMN–heme intraprotein electron transfer (IET). Although the amplitude of the IET kinetic traces was decreased two- to three-fold, the AR deletion did not change the rate constant for the calmodulin-controlled IET. This suggests that the rate-limiting conversion of the electron-accepting state to a new electron-donating (output) state does not involve interactions with the AR insert, but that AR may stabilize the output state once it is formed.

Abbreviations: NOS, nitric oxide synthase, nNOS, neuronal NOS, CaM, calmodulin, AR, autoregulatory insert within the FMN domain of nNOS, nNOS-AR, AR-deletion mutant of nNOS, oxyFMN, two-domain NOS construct in which only the heme-containing oxygenase and FMN domains are present, nNOSoxy, nNOS oxygenase construct, IET, intraprotein electron transfer, dRF, 5-deazariboflavin, H₄B, 6R-5,6,7,8-tetrahydrobiopterin

Keywords: Electron transfer, Nitric oxide synthase, Kinetic