Abstract 148: Superoxide Promotes Reversible Uncoupling Of Inducible Nitric Oxide Synthase, While Peroxynitrite Induces Irreversible And Complete Enzyme Inactivation
Inducible nitric oxide synthase (iNOS) is present in the post-ischemic heart, and plays an important role in the pathogenesis of injury and remodeling. Oxidants formed during reperfusion injury are known to alter NOS function. In this study we compare and contrast how two biologically relevant oxidants, peroxynitrite (ONOO−) and superoxide (O2 −), alter iNOS function and define the mechanisms involved for each. Nitric oxide (NO) generation rate of purified iNOS was quantified by the methemoglobin formation, O2−. generation was measured using EPR spin-trapping, and the quaternary structure of iNOS by size-exclusion FPLC. Tetrahydrobiopterin (BH4)-replete iNOS (1 μM) was exposed to O2−., and ONOO− in concentrations from 0.01 μM to 500μM and NO production was measured with and without addition of excess BH4. ONOO −(50μM) decreased NO production to 19% ± 0.17 and excess BH4 only partially restored activity to 48% ± 3.4. O2−. (50 μM) reduced NO production to 42% ± 1.2 and the excess BH4 completely restored activity to 100% ± 3.1. ONOO− exerted the highest decrease in NO production rate (96% ± 1.3 activity loss at 500 μM ONOO−) and no activity was restored by excess BH4. In contrast, O2 −. induced a significant decrease (90% ± 1.2 activity loss at 500 μM O2−.), however excess BH4 restored the iNOS activity back to 69% ± 5.6.O2−. exposure enhanced O2 −. production from iNOS (up to 155% ± 0.72 at 50 μM O2−.), while ONOO−at higher concentrations (>20μM) reduced O2 −. production (10% ± 3.2 decrease at 50 μM ONOO−). We also found that incubation with ONOO− and O2 −. induced iNOS monomerization. In conclusion, both ONOO− and O2 −. decrease iNOS NO production in a dose dependent manner. Adding excess BH4 only partially restore the loss of NO production induced by ONOO −, but almost completely restore the loss of NO production induced by O2 −.. Therefore, O2 −. promotes the uncoupling of iNOS and elevates O2−. production by oxidizing the protein bound BH4 with subsequent formation of NOS monomer in a reversible fashion. Conversely, ONOO− induces irreversible enzyme inactivation and decreases both NO and O2−. production. Although oxidation of BH4 is involved in the observed ONOO− induced loss of iNOS activity, there are other causes, including oxidation of critical amino acid residues.