The Characteristics and Regulatory Mechanisms of Superoxide Generation from eNOS Reductase Domain

In addition to superoxide (O₂ ^.-) generation from nitric oxide synthase (NOS) oxygenase domain, a new O₂ ^.- generation site has been identified in the reductase domain of inducible NOS (iNOS) and neuronal NOS (nNOS). Cysteine S-glutathionylation in eNOS reductase domain also induces O₂ ^.- generation from eNOS reductase domain. However, the characteristics and regulatory mechanism of the O₂ ^.- generation from NOS reductase domain remain unclear. We cloned and purified the wild type bovine eNOS (WT eNOS), a mutant of Serine 1179 replaced with aspartic acid eNOS (S1179D eNOS), which mimics the negative charge caused by phosphorylationand truncated eNOS reductase domain (eNOS RD). Both WT eNOS and S1179D eNOS generated significant amount of O₂ ^.- in the absence of BH4 and L-arginine. The capacity of O₂ ^.- generation from S1179D eNOS was significantly higher than that of WT eNOS (1.74:1). O₂ ^.- generation from both WT eNOS and S1179D eNOS were not completely inhibited by 100nM tetrahydrobiopterin(BH4). This BH4 un-inhibited O₂ ^.- generation from eNOS was blocked by 10mM flavoprotein inhibitor, diphenyleneiodonium (DPI). Purified eNOS reductase domain protein confirmed that this BH4 un-inhibited O₂ ^.- generation originates at the FMN or FAD/NADPH binding site of eNOS reductase domain. DEPMPO-OOH adduct EPR signals and NADPH consumptions analyses showed that O₂ ^.- generation from eNOS reductase domain was regulated by Serine 1179 phosphorylation and DPI, but not by L-arginine, BH4 or calmodulin (CaM). In addition to the heme center of eNOS oxygenase domain, we confirmed another O₂ ^.- generation site in the eNOS reductase domain and characterized its regulatory properties.