It is clear that the formation of S-nitrosothiols occurs as a product of the formation of NO and that a certain amount of S-nitrosation accompanies NO formation. These levels of S-nitrosothiols have been prescribed functions and activities that are crucial to the regulation of many important physiological and pathophysiological events. If S-nitrosation does indeed play such a role, then it would seem essential that the formation and decay of these species should be regulated and controlled, and not left to the random NO chemistry just described. There is currently very little evidence for such controlling mechanisms. CP has been reported to be a nitrosothiol synthase in plasma as just discussed. The idea that superoxide dismutase represents a catalyst for the formation of S-nitrosohemoglobin (38
), and that hemoglobin undergoes self-nitrosation during oxygenation deoxygenation cycles, is controversial (47
). The lack of a specific and dedicated S-nitrosothiol synthetic machinery is a major weakness of the concept that S-nitrosothiol synthesis is a targeted and directed signaling process (64
). In the absence of specific “kinase” analogs, the targeting of S-nitrosation would be determined by mechanisms based on thiol pKa, hydrophobicity and localization of source and target. Each of these concepts will be discussed in detail.
Thiol pKa is the primary determinant of thiol reactivity. Although most protein thiols possess a pKa within the range of 8–9 (14
), some thiols exhibit proton dissociation constants that are several orders of magnitude outside this range, from pKa values of ~4–11. Thiolate is one of the most reactive protein functional groups and is a much stronger nucleophile than a thiol. Thus, it will generally react many times faster with biologically relevant oxidants and electrophiles. Proteins that contain thiols with particularly low pKa values are more susceptible to oxidative modification due to the higher proportion of ionization at physiological pH. Thiols are positioned in environments that engender a low pKa for a multiplicity of apparent reasons, including catalytic activity [e.g.
, thiol disulfide exchange in thioredoxin (22
)], facilitation of substrate binding (30
), or for purposes of allosteric regulation (41
). It may well be the case that cysteines with low pKa are good candidates for protein S-nitrosation, but this should not be invoked as a specific targeting mechanism in the same way, for example, as a consensus motif for kinase-dependent phosphorylation (2
). The ability of low pKa thiols to respond to oxidants and nitrosating agents is more akin to a stress response in which a certain threshold of oxidant concentration results in enough modification of the thiol target to elicit a biological response. A classic example of this kind of mechanism is the Keap1 protein that dissociates from Nrf2 in response to the electrophilic and oxidative modification of key cysteine residues which allows Nrf2 to translocate to the nucleus and act as a regulator of transcription (61
). The difference in pKa of a cysteine residue is possibly one of the major reasons, in combination with steric constraints and solvent accessibility, why a purified protein will exhibit selective S-nitrosation of one or more thiols, in the presence of nitrosating agents. It is likely that thiols with low pKa values are critical to some aspect of protein function (e.g.
, active site cysteines); thus, S-nitrosation of these thiols can potentially have a functional consequence.
It has been postulated that thiols in hydrophobic regions of membranes or proteins are more prone to S-nitrosation (43
). Hydrophobicity enhances the rate of reaction between NO and oxygen by a significant factor (30–300-fold) due to the fact that both NO and oxygen are hydrophobic gasses and preferentially partition into hydrophobic phases (64
). As shown by Moller et al.
), the presence of low-density lipoprotein significantly increased the yield of GSNO formation from the NO donor PROLI/NO. However, these studies do not say, and in some cases explicitly warn against the idea (72
), that this would facilitate the S-nitrosation of thiols in hydrophobic pockets. The major reason that hydrophobic thiol nitrosation is unlikely is that thiol groups in aprotic regions will be protonated and, thus, will be poorly reactive with S-nitrosating agents. Recent studies looking at the S-nitrosation of transmembrane spanning model peptides clearly show dramatically decreased S-nitrosation the deeper the thiol is placed within a model membrane (102
). It may be that low pKa thiols in the vicinity of membranes will experience a higher steady state of S-nitrosating agents than cytosolic proteins at a distance from the membrane, but it is not currently clear, considering the kinetics and dynamics of S-nitrosation chemistry and the diffusivity of NO and derivative nitrosating species, how much of a localization effect this will have. Moller et al.
have estimated that if nitrogen dioxide is formed in a membrane, it could travel 40-membrane thicknesses in the presence of 5
). If GSH is the major target for nitrogen dioxide resulting in GSNO formation, the nitrosation envelope is then defined by the diffusivity and reactivity of GSNO with protein thiols. The work of Nudler and co-workers has been especially influential in suggesting that hydrophobic motifs of proteins may promote S-nitrosation (74
). These authors suggested that bovine serum albumin could catalyze thiol S-nitrosation by providing a hydrophobic sub-domain. These studies did not directly measure S-nitrosothiols but electrochemically measured NO release after the addition of large amounts of Cu2+
and assumed that this was quantitatively related to the level of S-nitrosothiol formed. Our studies using a more direct detection of S-nitrosothiols (high performance liquid chromatography [HPLC] and mercury-inhibitable tri-iodide-based chemiluminescence) have shown both bovine and human serum albumin as causing a small but significant decrease
in S-nitrosothiol yield (58
). The concept of hydrophobicity-targeted S-nitrosation can, therefore, be regarded as unproven and chemically unlikely.
It is possible that specificity is engendered by rates of degradation rather than by rates of formation, such that rapidly degraded S-nitrosothiols would have less influence than more resistant ones. Although limited evidence suggests that different S-nitrosothiols can have different biological lifetimes, there is little evidence for specific enzymatic denitrosating activities. Although GSH-dependent formaldehyde (dehydrogenase) catalyzes the decomposition of GSNO (53
) and metabolizes cellular S-nitrosothiols, it lacks activity toward protein targets. Thioredoxin and protein disulfide isomerase have been shown to catalyze protein denitrosation (6
), but it is not clear whether these enzymes have the specificity to influence selective pathways and, thus, modulate the direction of NO signaling. At minimum, these enzymes likely play a role in the modulation of the overall S-nitrosothiol level. Although more is known about S-nitrosothiol decomposition than about biosynthesis, much still needs to be understood about how these pathways influence NO-dependent signaling.
There has been significant recent discussion about the issue of targeting S-nitrosation by the co-localization of NO synthase (NOS) with the target thiols (23
). In addition, it has been shown that altering endothelial NOS (eNOS) localization can alter the distribution of fluorescent dye after the labeling of S-nitrosoproteins (50
). These studies used “switch” methods and so can be regarded as somewhat indirect; however, the fact that the localization of eNOS alters patterns of labeling gives rise to some intriguing possibilities concerning mechanism. Biological localization is easy to understand, as it relies on the recognition of specific protein structural motifs to bring, for example, a kinase in close proximity to its protein substrate to facilitate phosphorylation. This is poorly analogous to NO signaling, as NOS is not thought to be a catalyst of S-nitrosation but is rather the source of a necessary substrate. The localization of effects that depend on the chemical biology of reactive species is much harder to rationalize. The hydroxyl radical, with a half life in nanoseconds, has long been considered a local oxidant due to its inherently high reactivity with almost all biological molecules (27
). In this case, damage has to be local to the site of oxidant formation. Peroxynitrite, a highly reactive oxidant with a millisecond biological half life, is thought to be able to react across the diameter of a cell (76
). NO, in comparison, is extremely stable with a biological half life in seconds. It is clear that NO itself cannot be localized at the sub-cellular level. The evidence for this is somewhat overwhelming, as extracellular oxyhemoglobin (oxyHb) is a potent vasoconstrictor and antagonist of EDRF (29
). The only plausible mechanism for this activity is the ability of oxyHb in the lumen of a blood vessel to destroy NO. This means that NO should be freely and widely diffusible, and NO-dependent activities can be inhibited by an extracellular scavenger (59
). We have previously published that the formation of S-nitrosothiols in activated macrophages can be antagonized by oxyHb (105
), as can the ability of extracellular S-nitrosothiols to activate sGC (82
). In fact, there is a long tradition of antagonizing NO-dependent effects in organs or cell culture systems by oxyHb, and increased plasma hemoglobin, and consequent EDRF antagonism, has been thought of as a pathological event in hemolytic disorders (81
). The packaging of hemoglobin in red cells has been shown to reduce its ability to scavenge endothelium-derived NO (66
). All this points to the fact that NO is not locally constrained at the sub-cellular level, even when the target reacts with NO at near-diffusion controlled rates (i.e.
, sGC). This raises the paradox of how localized NO production can result in localized S-nitrosation (or perhaps more accurately protein thiol modification) in a hemoglobin-insensitive manner, as recently described (50
). One logical response to this paradox is that it is the localization of some unknown catalytic activity and not the localization of NO formation that is crucial to such targeting, and that eNOS somehow provides or attracts this activity. The problem with this explanation is that it makes S-nitrosation somewhat NO-concentration independent, in the same way that phosphorylation is somewhat ATP-concentration independent. There are currently no data to support such possibilities, and since the detection of localized S-nitrosothiol formation is necessarily indirect, these speculations may be premature.
In total, our knowledge of the mechanisms of S-nitrosothiol formation in vivo is tenuous. It is not clear that we need to invoke “unknown mechanisms” which explain the levels of S-nitrosothiol detected in vivo, and it may be that the currently established chemistry of NO is sufficient. However, in order to explain the specificity of S-nitrosation that is inherent in many S-nitrosothiol-dependent signaling pathways, novel and currently unknown targeted pathways of protein S-nitrosation need to be invoked.