S-nitrosylation of endothelial nitric oxide synthase is associated with monomerization and decreased enzyme activity.
Endothelial nitric oxide synthase (eNOS) is active only as a homodimer. Recent data has demonstrated that exogenous NO can act as an inhibitor of eNOS activity both in intact animals and vascular endothelial cells. However, the exact mechanism by which NO exerts its inhibitory action is unclear. Our initial experiments in bovine aortic endothelial cells indicated that exogenous NO decreased NOS activity with an associated decrease in eNOS dimer levels. We then undertook a series of studies to investigate the mechanism of dimer disruption. Exposure of purified human eNOS protein to NO donors or calcium-mediated activation of the enzyme resulted in a shift in eNOS from a predominantly dimeric to a predominantly monomeric enzyme. Further studies indicated that endogenous NOS activity or NO exposure caused S-nitrosylation of eNOS and that the presence of the thioredoxin and thioredoxin reductase system could significantly protect eNOS dimer levels and prevent the resultant monomerization and loss of activity. Further, exogenous NO treatment caused zinc tetrathiolate cluster destruction at the dimer interface. To further determine whether S-nitrosylation within this region could explain the effect of NO on eNOS, we purified a C99A eNOS mutant enzyme lacking the tetrathiolate cluster and analyzed its oligomeric state. This enzyme was predominantly monomeric, implicating a role for the tetrathiolate cluster in dimer maintenance and stability. Therefore, this study links the inhibitory action of NO with the destruction of zinc tetrathiolate cluster at the dimeric interface through S-nitrosylation of the cysteine residues.
Ravi
Kandasam
K
Department of Pediatrics, Northwestern University, Chicago, IL 60611-3008, USA.
Brennan
Lisa A
LA
Levic
Snezana
S
Ross
Patrick A
PA
Black
Stephen M
SM
D000818:Animals; D001011:Aorta; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D019281:Dimerization; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D009589:Nitrogen Oxides; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D013096:Spermine / Q000031:analogs & derivatives* / Q000494:pharmacology
D020030:Nitric Oxide Donors; D009589:Nitrogen Oxides; D011994:Recombinant Proteins; C091861:spermine nitric oxide complex; D013096:Spermine; D009569:Nitric Oxide; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D002118:Calcium