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 (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.
Chen
Weiguo
W
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.
Druhan
Lawrence J
LJ
Chen
Chun-An
CA
Hemann
Craig
C
Chen
Yeong-Renn
YR
Berka
Vladimir
V
Tsai
Ah-Lim
AL
Zweier
Jay L
JL
D001708:Biopterins / Q000031:analogs & derivatives* / Q000737:chemistry; D001880:Boranes / Q000737:chemistry; D004795:Enzyme Stability; D006418:Heme / Q000737:chemistry*; D006427:Hemin / Q000737:chemistry; D006801:Humans; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D030421:Peroxynitrous Acid / Q000737:chemistry*; D011485:Protein Binding; D055503:Protein Multimerization; D015032:Zinc / Q000737:chemistry
D001880:Boranes; D030421:Peroxynitrous Acid; D001708:Biopterins; D006418:Heme; D006427:Hemin; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; C003402:sapropterin; D015032:Zinc