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

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 (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.

Figure 1

Inactivation of eNOS activity by ONOO⁻ and the protective effect of BH₄. Purified human eNOS (0.1 μg/μL) in 50 mM HEPES, pH 7.4, was treated with the indicated concentrations of ONOO⁻. After incubation (45 min on ice) eNOS activity was measured as described in the Materials and Methods. The values of eNOS activity presented are obtained from the average of six independent assays (*, P<0.05).

Figure 2

ONOO⁻-induced eNOS dimer destabilization and the protective effects of BH₄. (A) Purified human eNOS (0.1 μg/μL) in 50 mM HEPES, pH 7.4, was preincubated with L-arginine (100 μM) or BH₄ (100 μM) at 0 °C for 30 min. The enzyme was treated with indicated concentration of ONOO⁻ for 45 min at 0 °C. The entire mixture was then subjected to LT-PAGE, and the protein bands were visualized by Coomassie Blue staining. (B) The ratio of eNOS dimer/monomer in (A) was quantitated by digital densitometry as described in the Materials and Methods. The data analyses were obtained from the average of six independent experiments.

Figure 3

ONOO⁻-induced oxidation of BH₄ in eNOS or free BH₄ as measured by HPLC with electrochemical detection. Purified eNOS (0.15 μg/μL) or free BH₄ (1 μM) was incubated in 100 μL of 50 mM HEPES, pH 7.4, with different concentrations of ONOO⁻ as indicated for 45 min. 80 μl of BH₄ extraction buffer was added into the solution and centrifuged for 15 min at 16,000 × g. The supernatant was filtered and aliquots (25 μl) of the filtrate were subjected to HPLC analysis.

Figure 4

ONOO⁻-induced changes in the UV/VIS spectra of eNOS and pure heme. (A) eNOS (0.24 μg/μL) in 50 mM HEPES, pH 7.4, was treated with various concentrations of ONOO⁻, incubated for 10 min at RT, and the UV/VIS spectra recorded. (B) same as (A) except that the buffer included 5% pyridine and the eNOS was replaced with pure iron protoporphyrin (2 μM).

Figure 5

Detection of heme release from ONOO⁻-treated eNOS by heme staining. (A) Purified human eNOS (0.1 μg/μL) was incubated in HEPES, pH 7.4, with the indicated concentration of ONOO⁻ for 45 min. The samples were subjected to LT-PAGE, and heme staining as described in Materials and Methods. (B) The heme content was quantitated by digital densitometry as described in Materials and Methods, and represented as the percent of the total eNOS heme detected in either the monomer or dimer.

Figure 6

Effect of TPEN, Zn²⁺, and BH₄ on eNOS dimerization. (A) Purified human eNOS (0.1 μg/μL) in 50 mM HEPES, pH 7.4, was treated with various concentrations of TPEN for 45 min in the presence or absence of 100 μM Zn²⁺. The mixture was subjected to LT-PAGE and the protein bands were visualized by coomassie blue staining. (B) Same as (A), except the eNOS was preincubated with or without BH₄ (100 μM) prior to addition of TPEN and Zn²⁺. The ratios of eNOS dimer/monomer were quantitated by digital densitometry as described in Materials and Methods.

Figure 7

Effects of BH₄ post-addition on the restoration of eNOS dimer stability and the enzymatic activity of eNOS. (A) eNOS (0.2 μg/μL) in HEPES, pH 7.4, was treated with 100 μM ONOO⁻. BH₄, L-arginine, or both were then added to the ONOO⁻-treated eNOS. The mixtures were incubated for 30 min, and then subjected to LT-PAGE and staining with coomassie blue. (B) Samples were the same as (A), except that aliquots of each mixture were withdrawn for measurement of eNOS activity as described in Materials and Methods.

Figure 7

Effects of BH₄ post-addition on the restoration of eNOS dimer stability and the enzymatic activity of eNOS. (A) eNOS (0.2 μg/μL) in HEPES, pH 7.4, was treated with 100 μM ONOO⁻. BH₄, L-arginine, or both were then added to the ONOO⁻-treated eNOS. The mixtures were incubated for 30 min, and then subjected to LT-PAGE and staining with coomassie blue. (B) Samples were the same as (A), except that aliquots of each mixture were withdrawn for measurement of eNOS activity as described in Materials and Methods.