Peroxynitrite Induces Destruction of the Tetrahydrobiopterin and Heme in Endothelial Nitric Oxide Synthase: Transition from Reversible to Irreversible Enzyme Inhibition
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- 作者：Weiguo Chen ; Lawrence J. Druhan ; Chun-An Chen ; Craig Hemann ; Yeong-Renn Chen ; Vladimir Berka ; Ah-Lim Tsai ; Jay L. Zweier
- 刊名：Biochemistry
- 出版年：2010
- 出版时间：April 13, 2010
- 年：2010
- 卷：49
- 期：14
- 页码：3129-3137
- 全文大小：891K
- 年卷期：v.49,no.14(April 13, 2010)
- ISSN：1520-4995
文摘
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 (BH4); 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 BH4 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 BH4 decreased dimer destabilization and heme alteration. Addition of BH4 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 BH4 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 BH4 and irreversible destruction of the heme/heme center.