Peroxynitrite induces destruction of the tetrahydrobiopterin and heme in endothelial nitric oxide synthase

Transition from reversible to irreversible enzyme inhibition

Weiguo Chen, Lawrence J. Druhan, Chun An Chen, Craig Hemann, Yeong Renn Chen, Vladimir Berka, Ah Lim Tsai, Jay L. Zweier

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3129-3137
Number of pages9
JournalBiochemistry®
Volume49
Issue number14
DOIs
StatePublished - Apr 13 2010
Externally publishedYes

Fingerprint

Enzyme inhibition
Peroxynitrous Acid
Nitric Oxide Synthase Type III
Heme
Enzymes
Dimers
Zinc
Oxidation
sapropterin
Biopterin
Enzyme activity
Chelating Agents
Blood Vessels

ASJC Scopus subject areas

  • Biochemistry

Cite this

Peroxynitrite induces destruction of the tetrahydrobiopterin and heme in endothelial nitric oxide synthase : Transition from reversible to irreversible enzyme inhibition. / Chen, Weiguo; Druhan, Lawrence J.; Chen, Chun An; Hemann, Craig; Chen, Yeong Renn; Berka, Vladimir; Tsai, Ah Lim; Zweier, Jay L.

In: Biochemistry®, Vol. 49, No. 14, 13.04.2010, p. 3129-3137.

Research output: Contribution to journalArticle

Chen, Weiguo ; Druhan, Lawrence J. ; Chen, Chun An ; Hemann, Craig ; Chen, Yeong Renn ; Berka, Vladimir ; Tsai, Ah Lim ; Zweier, Jay L. / Peroxynitrite induces destruction of the tetrahydrobiopterin and heme in endothelial nitric oxide synthase : Transition from reversible to irreversible enzyme inhibition. In: Biochemistry®. 2010 ; Vol. 49, No. 14. pp. 3129-3137.
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T2 - Transition from reversible to irreversible enzyme inhibition

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AU - Druhan, Lawrence J.

AU - Chen, Chun An

AU - Hemann, Craig

AU - Chen, Yeong Renn

AU - Berka, Vladimir

AU - Tsai, Ah Lim

AU - Zweier, Jay L.

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N2 - 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.

AB - 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.

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