Peroxynitrite induces destruction of the tetrahydrobiopterin and heme in endothelial nitric oxide synthase: Transition from reversible to irreversible enzyme inhibition

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.