Nitric-oxide Synthase Forms N-NO-pterin and S-NO-Cys

Abstract

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. Nitric oxide (NO)4 levels are regulated in vivo at the level of synthesis by three closely related forms of nitric-oxide synthase (NOS): inducible (iNOS), endothelial (eNOS), and neuronal (nNOS) (1). All three NOS enzymes are active only as homodimers and contain two modules: the catalytic oxygenase (NOS ox ) and the electron-supplying reductase (NOS red ) (2). The NOS ox dimer contains two catalytic sites at which the substrate L-arginine binds above the heme (3, 4). The symmetrical NOS ox dimer interface is stabilized by a bridging ZnS 4 cluster and two predominantly buried tetrahydrobiopterin (H 4 B or (6R)-5,6,7,8-tetrahydro-L-biopterin) cofactors, each hydrogen-bonded to one heme (4 -7). Critical differences among the three isozymes include location, regulatory mechanisms, dimer stability, and the amount of NO that is produced (8 -10). iNOS rapidly generates large toxic bursts of NO in vivo and has fewer known mechanisms for halting NO production, whereas eNOS and nNOS produce less NO, are calcium-regulated and contain auto-inhibitory elements (9,11,12).Biological signaling by NO is orchestrated by three interconverting redox-active forms: the free radical (NO ⅐ ), the nitroxide anion (NO Ϫ ), and the nitrosonium cation (NO ϩ ) (13). NO ⅐ binds to metal sites in proteins, while NO ϩ reacts with cysteinyl sulfurs forming S-nitrosylated derivatives (14). S-Nitrosylation is a reversible post-translational modification implicated in protein regulation and signaling within intact cells (15-17). As the major...

S-Nitrosylation signaling regulates cellular protein interactions

“…Note that different mechanisms have been identified by which NOS’s can be S-nitrosothiol synthases. In the case of iNOS, for example, the formation of one R-N-NO intermediate on the pterin has been identified near a GSH binding site (30). …”

Mechanism and Kinetics of Inducible Nitric Oxide Synthase Auto-S-nitrosation and Inactivation

“…While iNOS was previously shown to be S- nitrosated by exogenously added NO (8) and endogenously generated NO at the heme cofactor (30), the timescale of auto- S -nitrosation were not investigated. Here, iNOS was rapidly S -nitrosated by NO generated during the catalytic reaction (Figure 1A), and the level of iNOS auto- S -nitrosation peaked at ~30 minutes and did not increase at 60 minutes.…”

Nitric Oxide Signaling in Biology

“…In mice, deficiency in iNOS results in reduced atherosclerosis lesions (Detmers et al, 2000). Once iNOS is expressed, it produces large toxic bursts of NO for long periods of time (hours to days) (MacMicking et al, 1997;Rosenfeld et al, 2010). In addition, iNOS protein content in fully activated cells may be higher than the constitutive NOS content (MacMicking et al, 1997).…”

“…eNOS and iNOS are susceptible to NO-induced thiol modifications (Erwin et al, 2006;Rosenfeld et al, 2010). S-nitrosylation of NOS enzymes regulates the NO production suggesting a feedback mechanism to control its activity (Erwin et al, 2006;Rosenfeld et al, 2010). In endothelial cells PrSNO levels are increased under various conditions (Handy and Loscalzo, 2006).…”

“…However, the biochemical basis for this selectivity is not clear. eNOS and iNOS are susceptible to NO-induced thiol modifications (Erwin et al, 2006;Rosenfeld et al, 2010). S-nitrosylation of NOS enzymes regulates the NO production suggesting a feedback mechanism to control its activity (Erwin et al, 2006;Rosenfeld et al, 2010).…”