Neuronal nitric oxide synthase (nNOS) generates superoxide, particularly at sub-optimal l-arginine (l-Arg) substrate concentrations. Heat shock protein 90 (Hsp90) was reported to inhibit superoxide generation from nNOS protein. However, commercially available Hsp90 product from bovine brain tissues with unspecified Hsp90α and Hsp90β contents and an undefined Hsp90 protein oligomeric state was utilized. These two Hsp90s can have opposite effect on superoxide production by NOS. Importantly, emerging evidence indicates that nNOS splice variants are involved in different biological functions by functioning distinctly in redox signaling. In the present work, purified recombinant human Hsp90α, in its native dimeric state, was used in electron paramagnetic resonance (EPR) spin trapping experiments to study the effects of Hsp90α on superoxide generation from nNOS splice variants nNOSμ and nNOSα. Human Hsp90α was found to significantly increase superoxide generation from nNOSμ and nNOSα proteins under l-Arg-depleted conditions and Hsp90α influenced superoxide production by nNOSμ and nNOSα at varying degrees. Imidazole suppressed the spin adduct signal, indicating that superoxide was produced at the heme site of nNOS in the presence of Hsp90α, whereas l-Arg repletion diminished superoxide production by the nNOS-Hsp90α. Moreover, NADPH consumption rate values exhibited a similar trend/difference as a function of Hsp90α and l-Arg. Together, these EPR spin trapping and NADPH oxidation kinetics results demonstrated noticeable Hsp90α-induced increases in superoxide production by nNOS and a distinguishable effect of Hsp90α on nNOSμ and nNOSα proteins.

神经元一氧化氮合酶 (nNOS) 产生超氧化物，特别是在次优的l-精氨酸 ( l-Arg) 底物浓度。据报道，热休克蛋白 90 (Hsp90) 可抑制 nNOS 蛋白产生的超氧化物。然而，使用了来自牛脑组织的市售 Hsp90 产品，其具有未指定的 Hsp90α 和 Hsp90β 含量以及未定义的 Hsp90 蛋白寡聚状态。这两个 Hsp90 对 NOS 产生的超氧化物有相反的影响。重要的是，新出现的证据表明，nNOS 剪接变体通过在氧化还原信号传导中的不同功能参与不同的生物学功能。在目前的工作中，纯化的重组人 Hsp90α 以其天然二聚体状态用于电子顺磁共振 (EPR) 自旋捕获实验，以研究 Hsp90α 对 nNOS 剪接变体 nNOSμ 和 nNOSα 产生超氧化物的影响。l -Arg 耗尽的条件和 Hsp90α 在不同程度上影响了 nNOSμ 和 nNOSα 产生的超氧化物。咪唑抑制自旋加合物信号，表明在 Hsp90α 存在下，nNOS 的血红素位点产生了超氧化物，而l -Arg 的补充减少了 nNOS-Hsp90α 产生的超氧化物。此外，作为 Hsp90α 和l -Arg的函数，NADPH 消耗率值表现出类似的趋势/差异。总之，这些 EPR 自旋捕获和 NADPH 氧化动力学结果表明 Hsp90α 诱导的 nNOS 产生的超氧化物显着增加，以及 Hsp90α 对 nNOSμ 和 nNOSα 蛋白的显着影响。