Mass spectroscopy and molecular modeling predict endothelial nitric oxide synthase dimer collapse by hydrogen peroxide through zinc tetrathiolate metal-binding site disruption.
Endothelial nitric oxide synthase (eNOS) is inhibited by hydrogen peroxide (H(2)O(2)), but the mechanism has not been determined. Thus, the purpose of this study was to delineate the mechanism by which H(2)O(2) inhibits eNOS activity. Using mass spectroscopy, we found that the tetrathiolate cysteine residues 94 and 99 were susceptible to oxidation by H(2)O(2). 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 H(2)O(2) caused disruption of the heNOS dimer and this was accompanied by zinc release and decreased NO generation. We also found that H(2)O(2) increased the oxidation of tetrahydrobiopterin (BH(4)) to dihydrobiopterin (BH(2)), whereas preincubation of heNOS with excess BH(4) prevented the destruction of zinc tetrathiolate and dimer collapse and preserved activity. Interestingly, we found that the dimmer-stabilizing effect of BH(4) is due to its ability to act as a catalase mimetic. Further, we confirmed that, in ovine aortic endothelial cells, H(2)O(2) could also induce dimer collapse and that increasing cellular BH(4) levels could maintain eNOS in its dimeric form and NO signaling when cells were challenged with H(2)O(2). This study links the inhibitory action of H(2)O(2) on heNOS through the destruction of zinc tetrathiolate metal-binding site and dimer collapse both in vitro and in vivo.
Fonseca
Fabio V
FV
Vascular Biology Center, Medical College of Georgia, Augusta, 30912, USA.
Ravi
Kandasamy
K
Wiseman
Dean
D
Tummala
Monorama
M
Harmon
Cynthia
C
Ryzhov
Victor
V
Fineman
Jeffrey R
JR
Black
Stephen M
SM
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000187:drug effects; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D003544:Cysteic Acid / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000096:biosynthesis; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects*; D020836:Protein Structure, Quaternary / Q000187:drug effects; D015032:Zinc / Q000378:metabolism*
D001708:Biopterins; D009569:Nitric Oxide; D003544:Cysteic Acid; D006861:Hydrogen Peroxide; D052250:Nitric Oxide Synthase Type III; C003402:sapropterin; D015032:Zinc; D003545:Cysteine