Abstract 148: Superoxide Promotes Reversible Uncoupling Of Inducible Nitric Oxide Synthase, While Peroxynitrite Induces Irreversible And Complete Enzyme Inactivation

Abstract

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 (O 2 − ), alter iNOS function and define the mechanisms involved for each. Nitric oxide (NO) generation rate of purified iNOS was quantified by the methemoglobin formation, O 2 −. generation was measured using EPR spin-trapping, and the quaternary structure of iNOS by size-exclusion FPLC. Tetrahydrobiopterin (BH 4 )-replete iNOS (1 μM) was exposed to O 2 −. , and ONOO − in concentrations from 0.01 μM to 500μM and NO production was measured with and without addition of excess BH 4 . ONOO − (50μM) decreased NO production to 19% ± 0.17 and excess BH 4 only partially restored activity to 48% ± 3.4. O 2 −. (50 μM) reduced NO production to 42% ± 1.2 and the excess BH 4 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 BH 4 . In contrast, O 2 −. induced a significant decrease (90% ± 1.2 activity loss at 500 μM O 2 −. ), however excess BH 4 restored the iNOS activity back to 69% ± 5.6.O 2 −. exposure enhanced O 2 −. production from iNOS (up to 155% ± 0.72 at 50 μM O 2 −. ), while ONOO − at higher concentrations (>20μM) reduced O 2 −. production (10% ± 3.2 decrease at 50 μM ONOO − ). We also found that incubation with ONOO − and O 2 −. induced iNOS monomerization. In conclusion, both ONOO − and O 2 −. decrease iNOS NO production in a dose dependent manner. Adding excess BH 4 only partially restore the loss of NO production induced by ONOO − , but almost completely restore the loss of NO production induced by O 2 −.. Therefore, O 2 −. promotes the uncoupling of iNOS and elevates O 2 −. production by oxidizing the protein bound BH 4 with subsequent formation of NOS monomer in a reversible fashion. Conversely, ONOO − induces irreversible enzyme inactivation and decreases both NO and O 2 −. production. Although oxidation of BH 4 is involved in the observed ONOO − induced loss of iNOS activity, there are other causes, including oxidation of critical amino acid residues.