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Monomerization of inducible nitric oxide synthase blocks superoxide as well as nitric oxide production from the enzyme
Author(s) -
Sun Jian,
Druhan Lawrence J.,
Zweier Jay L.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1146
Subject(s) - chemistry , nitric oxide , nitric oxide synthase , urea , superoxide , peroxynitrite , biochemistry , protein quaternary structure , citrulline , enzyme , superoxide dismutase , arginine , organic chemistry , amino acid , protein subunit , gene
Inducible nitric oxide synthase (iNOS) is present in the post‐ischemic heart, and plays an important role in the pathogenesis of injury and remodeling. NOS enzymes use NADPH to produce nitric oxide (NO · ) and citrulline from oxygen and arginine. In addition, the NOS enzymes can also transfer electrons from NADPH to oxygen, producing superoxide (O 2 −· ). In all the NOS enzymes, the production of NO · by iNOS requires a dimeric quarternary structure. Previous work has indicated that under conditions of oxidative stress, the NOS quaternary structure is altered from dimer to monomer, and thus production of NO · is abated. However, there is a lack of knowledge regarding the potential of the iNOS monomer for the production of O 2 −· . To understand how the biologically relevant iNOS monomer functions, we used urea to induce monomerization and determine how this alters NO · and O 2 −· production. The NO · generation rate of purified iNOS was quantified by the methemoglobin formation assay, the O 2 −· generation rate was measured using EPR spin‐trapping, and the quaternary structure of iNOS was monitored by size‐exclusion FPLC. iNOS was pre‐exposed to urea in concentrations from 0.1M to 5M. iNOS mostly (>90%) presents as dimer at low urea concentration (≤1M urea). Both NO · and O 2 −· production from iNOS slightly increased with increasing of urea concentration (0.1M ~ 1M urea). iNOS dissociates into monomer at higher urea concentrations, with the monomer percentage significantly increased in the 2M ~ 5M urea range. NO · and O 2 −· production decreased proportional to the dissociation of iNOS dimer. In conclusion, the homodimer is the active form of iNOS and is essential for both NO · and O 2 −· production. As such iNOS monomerization appears to lead to complete inactivation of enzyme function.