Considerable evidence indicates that NO biology involves a family of NO-related molecules and that S-nitrosothiols (SNOs) are central to signal transduction and host defence. It is unknown, however, how cells switch off the signals or protect themselves from the SNOs produced for defence purposes. Here we have purified a single activity from Escherichia coli, Saccharomyces cerevisiae and mouse macrophages that metabolizes S-nitrosoglutathione (GSNO), and show that it is the glutathione-dependent formaldehyde dehydrogenase. Although the enzyme is highly specific for GSNO, it controls intracellular levels of both GSNO and S-nitrosylated proteins. Such 'GSNO reductase' activity is widely distributed in mammals. Deleting the reductase gene in yeast and mice abolishes the GSNO-consuming activity, and increases the cellular quantity of both GSNO and protein SNO. Furthermore, mutant yeast cells show increased susceptibility to a nitrosative challenge, whereas their resistance to oxidative stress is unimpaired. We conclude that GSNO reductase is evolutionarily conserved from bacteria to humans, is critical for SNO homeostasis, and protects against nitrosative stress.

Only a few intracellular S-nitrosylated proteins have been identified, and it is unknown if protein S-nitrosylation/denitrosylation is a component of signal transduction cascades. Caspase-3 zymogens were found to be S-nitrosylated on their catalytic-site cysteine in unstimulated human cell lines and denitrosylated upon activation of the Fas apoptotic pathway. Decreased caspase-3 S-nitrosylation was associated with an increase in intracellular caspase activity. Fas therefore activates caspase-3 not only by inducing the cleavage of the caspase zymogen to its active subunits, but also by stimulating the denitrosylation of its active-site thiol. Protein S-nitrosylation/denitrosylation can thus serve as a regulatory process in signal transduction pathways.

Nitric oxide (NO) biology has focused on the tightly regulated enzymatic mechanism that transforms Larginine into a family of molecules, which serve both signaling and defense functions. However, very little is known of the pathways that metabolize these molecules or turn off the signals. The paradigm is well exemplified in bacteria where S-nitrosothiols (SNO)-compounds identified with antimicrobial activities of NO synthase-elicit responses that mediate bacterial resistance by unknown mechanisms. Here we show that Escherichia coli possess both constitutive and inducible elements for SNO metabolism. Constitutive enzyme(s) cleave SNO to NO whereas bacterial hemoglobin, a widely distributed f lavohemoglobin of poorly understood function, is central to the inducible response. Remarkably, the protein has evolved a novel heme-detoxification mechanism for NO. Specifically, the heme serves a dioxygenase function that produces mainly nitrate. These studies thus provide new insights into SNO and NO metabolism and identify enzymes with reactions that were thought to occur only by chemical means. Our results also emphasize that the reactions of SNO and NO with hemoglobins are evolutionary conserved, but have been adapted for cell-specific function.Three major nitric-oxide synthases (NOSs) and a growing list of alternatively spliced or otherwise modified NOS isoforms regulate the transformation of L-arginine into a family of molecules that are involved in numerous biological processes (1). This wide variety of effects is the reflection of a basic signaling mechanism that is utilized by virtually all mammalian cells and many lower organisms. Specifically, NO groups are introduced into thiol-and transition metal-containing proteins, thereby altering their properties and functions. Among the target proteins known to be physiologically modified by NOSs (or their products) are several classes of ion channels (2, 3), ras protein (4), transcription factors (5-7), multiple enzymes (7,8), and hemoglobin (9). Potential NO binding͞ reaction sequences have been identified in some of these target proteins (7). Additional levels of regulation are provided by compartmentalization of the modifying enzymes, by control of NOS substrate or cofactor availability (which may influence product identity), and by the reactivities of small NO-donating molecules that can add NO groups (7,10,11).On the other hand, much less is known of the mechanism(s) that switch off NO signals or detach NO groups from proteins. For example, (S)NO groups in proteins are probably in equilibrium with low-mass SNOs, but the position of the equilibrium in cells and its contribution to NO group removal is not clear. An added peculiarity of redox systems is the dependency on metabolic enzymes, such as superoxide dismutase (SOD) and catalase, that prevent fortuitous damage by oxidant-signaling molecules (12). These enzymes are widely distributed in all cells and are induced by oxidative stresses. By analogy, little is known of the metabolic pathways that limit the r...