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Endothelial nitric oxide synthase (eNOS) is inhibited by hydrogen peroxide (H2O2), but the mechanism has not been determined. Thus, the purpose of this study was to delineate the mechanism by which H2O2 inhibits eNOS activity. Using mass spectroscopy, we found that the tetrathiolate cysteine residues 94 and 99 were susceptible to oxidation by H2O2. Molecular modeling predicted that these cysteic acid modifications would disrupt the van der Waals interactions and the hydrogen bonding network mediated by the tetrathiolate cysteines 94 and 99 resulting in changes in quaternary structure, zinc release, and dimer collapse. Using recombinant human eNOS (heNOS) to test the predictions of the molecular modeling we found that H2O2 caused disruption of the heNOS dimer and this was accompanied by zinc release and decreased NO generation. We also found that H2O2 increased the oxidation of tetrahydrobiopterin (BH4) to dihydrobiopterin (BH2), whereas preincubation of heNOS with excess BH4 prevented the destruction of zinc tetrathiolate and dimer collapse and preserved activity. Interestingly, we found that the dimmer-stabilizing effect of BH4 is due to its ability to act as a catalase mimetic. Further, we confirmed that, in ovine aortic endothelial cells, H2O2 could also induce dimer collapse and that increasing cellular BH4 levels could maintain eNOS in its dimeric form and NO signaling when cells were challenged with H2O2. This study links the inhibitory action of H2O2 on heNOS through the destruction of zinc tetrathiolate metal-binding site and dimer collapse both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

S-nitrosylation, or the replacement of the hydrogen atom in the thiol group of cysteine residues by a –NO moiety, is a physiologically important posttranslational modification. In our previous work we have shown that S-nitrosylation is involved in the disruption of the endothelial nitric oxide synthase (eNOS) dimer and that this involves the disruption of the zinc (Zn) tetrathiolate cluster due to the S-nitrosylation of Cysteine 98. However, human eNOS contains 28 other cysteine residues whose potential to undergo S-nitrosylation has not been determined. Thus, the goal of this study was to identify the cysteine residues within eNOS that are susceptible to S-nitrosylation in vitro. To accomplish this, we utilized a modified biotin switch assay. Our modification included the tryptic digestion of the S-nitrosylated eNOS protein to allow the isolation of S-nitrosylated peptides for further identification by mass spectrometry. Our data indicate that multiple cysteine residues are capable of undergoing S-nitrosylation in the presence of an excess of a nitrosylating agent. All these cysteine residues identified were found to be located on the surface of the protein according to the available X-ray structure of the oxygenase domain of eNOS. Among those identified were Cys 93 and 98, the residues involved in the formation of the eNOS dimer through a Zn tetrathiolate cluster. In addition, cysteine residues within the reductase domain were identified as undergoing S-nitrosylation. We identified cysteines 660, 801, and 1113 as capable of undergoing S-nitrosylation. These cysteines are located within regions known to bind flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide (NADPH) although from our studies their functional significance is unclear. Finally we identified cysteines 852, 975/990, and 1047/1049 as being susceptible to S-nitrosylation. These cysteines are located in regions of eNOS that have not been implicated in any known biochemical functions and the significance of their S-nitrosylation is not clear from this study. Thus, our data indicate that the eNOS protein can be S-nitrosylated at multiple sites other than within the Zn tetrathiolate cluster, suggesting that S-nitrosylation may regulate eNOS function in ways other than simply by inducing dimer collapse. [ABSTRACT FROM AUTHOR]