Nitric-oxide synthase forms N-NO-pterin and S-NO-cys: implications for activity, allostery, and regulation.
Inducible nitric-oxide synthase (iNOS) produces biologically stressful levels of nitric oxide (NO) as a potent mediator of cellular cytotoxicity or signaling. Yet, how this nitrosative stress affects iNOS function in vivo is poorly understood. Here we define two specific non-heme iNOS nitrosation sites discovered by combining UV-visible spectroscopy, chemiluminescence, mass spectrometry, and x-ray crystallography. We detected auto-S-nitrosylation during enzymatic turnover by using chemiluminescence. Selective S-nitrosylation of the ZnS(4) site, which bridges the dimer interface, promoted a dimer-destabilizing order-to-disorder transition. The nitrosated iNOS crystal structure revealed an unexpected N-NO modification on the pterin cofactor. Furthermore, the structurally defined N-NO moiety is solvent-exposed and available to transfer NO to a partner. We investigated glutathione (GSH) as a potential transnitrosation partner because the intracellular GSH concentration is high and NOS can form S-nitrosoglutathione. Our computational results predicted a GSH binding site adjacent to the N-NO-pterin. Moreover, we detected GSH binding to iNOS with saturation transfer difference NMR spectroscopy. Collectively, these observations resolve previous paradoxes regarding this uncommon pterin cofactor in NOS and suggest means for regulating iNOS activity via N-NO-pterin and S-NO-Cys modifications. The iNOS self-nitrosation characterized here appears appropriate to help control NO production in response to cellular conditions.
Rosenfeld
Robin J
RJ
Department of Molecular Biology, The Skaggs Institute for Chemical Biology, La Jolla, California 92037, USA.
Bonaventura
Joseph
J
Szymczyna
Blair R
BR
MacCoss
Michael J
MJ
Arvai
Andrew S
AS
Yates
John R
JR
Tainer
John A
JA
Getzoff
Elizabeth D
ED
D000494:Allosteric Regulation / Q000502:physiology; D000818:Animals; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000737:chemistry* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D055503:Protein Multimerization / Q000502:physiology*; D020836:Protein Structure, Quaternary; D011622:Pterins / Q000737:chemistry* / Q000378:metabolism
D011622:Pterins; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D005978:Glutathione; D003545:Cysteine