Regulation of the Monomer-Dimer Equilibrium in Inducible Nitric-oxide Synthase by Nitric Oxide

• Published in: The Journal of biological chemistry Vol. 281; no. 12; pp. 8197 - 8204
• Main Authors: Li, David, Hayden, Eric Y., Panda, Koustubh, Stuehr, Dennis J., Deng, Haiteng, Rousseau, Denis L., Yeh, Syun-Ru
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 24.03.2006; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Motifs; Animals; Arginine - chemistry; Biopterins - analogs & derivatives; Biopterins - chemistry; Chelating Agents - pharmacology; Chromatography; Cysteine - chemistry; Dimerization; Disulfides - chemistry; Escherichia coli - metabolism; Heme - chemistry; Iron - chemistry; Mass Spectrometry; Mice; Models, Chemical; Models, Molecular; Molecular Conformation; Nitric Oxide - chemistry; Nitric Oxide - metabolism; Nitric Oxide Synthase Type II - chemistry; Nitric Oxide Synthase Type II - metabolism; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Spectrophotometry; Spectrum Analysis, Raman; Structure-Activity Relationship; Sulfhydryl Compounds - chemistry; Time Factors; Urea - pharmacology; Zinc - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M507328200

The oxygenase domain of inducible nitric-oxide synthase exists as a functional tight homodimer in the presence of the substrate l-arginine and the cofactor tetrahydrobiopterin (H4B). In the absence of H4B, the enzyme is a mixture of monomer and loose dimer. We show that exposure of H4B-free enzyme to NO induces dissociation of the loose dimer into monomers in a reaction that follows single exponential decay kinetics with a lifetime of ∼300 min. It is followed by a faster autoreduction reaction of the heme iron with a lifetime of ∼30 min and the concurrent breakage of the proximal iron-thiolate bond, forming a five-coordinate NO-bound ferrous species. Mass spectrometry revealed that the NO-induced monomerization is associated with intramolecular disulfide bond formation between Cys104 and Cys109, located in the zinc-binding motif. The regulatory effect of NO as a dimer inhibitor is discussed in the context of the structure/function relationships of this enzyme.