Davis Heart and Lung Research Institute, Division of Cardiovascular Medicine, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, Ohio 43210, USA.
Biochemistry. 2010 Apr 13;49(14):3129-37. doi: 10.1021/bi9016632.
Endothelial nitric oxide synthase (eNOS) is an important regulator of vascular and cardiac function. Peroxynitrite (ONOO(-)) inactivates eNOS, but questions remain regarding the mechanisms of this process. It has been reported that inactivation is due to oxidation of the eNOS zinc-thiolate cluster, rather than the cofactor tetrahydrobiopterin (BH(4)); however, this remains highly controversial. Therefore, we investigated the mechanisms of ONOO(-)-induced eNOS dysfunction and their dose dependence. Exposure of human eNOS to ONOO(-) resulted in a dose-dependent loss of activity with a marked destabilization of the eNOS dimer. HPLC analysis indicated that both free and eNOS-bound BH(4) were oxidized during exposure to ONOO(-); however, full oxidation of protein-bound biopterin required higher ONOO(-) levels. Additionally, ONOO(-) triggered changes in the UV/visible spectrum and heme content of the enzyme. Preincubation of eNOS with BH(4) decreased dimer destabilization and heme alteration. Addition of BH(4) to the ONOO(-)-destabilized eNOS dimer only partially rescued enzyme function. In contrast to ONOO(-) treatment, incubation with the zinc chelator TPEN with removal of enzyme-bound zinc did not change the eNOS activity or stability of the SDS-resistant eNOS dimer, demonstrating that the dimer stabilization induced by BH(4) does not require zinc occupancy of the zinc-thiolate cluster. While ONOO(-) treatment was observed to induce loss of Zn binding, this cannot account for the loss of enzyme activity. Therefore, ONOO(-)-induced eNOS inactivation is primarily due to oxidation of BH(4) and irreversible destruction of the heme/heme center.
内皮型一氧化氮合酶（eNOS）是血管和心脏功能的重要调节剂。过氧亚硝酸盐（ONOO(-)）使 eNOS 失活，但关于这个过程的机制仍存在疑问。据报道，失活是由于 eNOS 的锌硫醇簇被氧化，而不是辅助因子四氢生物蝶呤（BH(4)）；然而，这仍然存在很大的争议。因此，我们研究了 ONOO(-)诱导的 eNOS 功能障碍及其剂量依赖性的机制。将人 eNOS 暴露于 ONOO(-)中，会导致活性呈剂量依赖性丧失，并且 eNOS 二聚体明显不稳定。HPLC 分析表明，在暴露于 ONOO(-)期间，游离和与 eNOS 结合的 BH(4)都被氧化；然而，蛋白质结合的生物蝶呤完全氧化需要更高的 ONOO(-)水平。此外，ONOO(-)触发了酶的紫外/可见光谱和血红素含量的变化。将 eNOS 与 BH(4)预孵育可减少二聚体的不稳定和血红素的改变。将 BH(4)添加到 ONOO(-)破坏的 eNOS 二聚体中仅部分恢复了酶的功能。与 ONOO(-)处理相反，用锌螯合剂 TPEN 孵育并去除酶结合的锌不会改变 eNOS 的活性或 SDS 抗性 eNOS 二聚体的稳定性，这表明 BH(4)诱导的二聚体稳定不需要锌硫醇簇的锌占据。虽然观察到 ONOO(-)处理会诱导 Zn 结合的丧失，但这不能解释酶活性的丧失。因此，ONOO(-)诱导的 eNOS 失活主要是由于 BH(4)的氧化和血红素/血红素中心的不可逆破坏。
