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.

Interactions of nitric oxide (NO) with hemoglobin (Hb) could regulate the uptake and delivery of oxygen (O(2)) by subserving the classical physiological responses of hypoxic vasodilation and hyperoxic vasconstriction in the human respiratory cycle. Here we show that in in vitro and ex vivo systems as well as healthy adults alternately exposed to hypoxia or hyperoxia (to dilate or constrict pulmonary and systemic arteries in vivo), binding of NO to hemes (FeNO) and thiols (SNO) of Hb varies as a function of HbO(2) saturation (FeO(2)). Moreover, we show that red blood cell (RBC)/SNO-mediated vasodilator activity is inversely proportional to FeO(2) over a wide range, whereas RBC-induced vasoconstriction correlates directly with FeO(2). Thus, native RBCs respond to changes in oxygen tension (pO2) with graded vasodilator and vasoconstrictor activity, which emulates the human physiological response subserving O(2) uptake and delivery. The ability to monitor and manipulate blood levels of NO, in conjunction with O(2) and carbon dioxide, may therefore prove useful in the diagnosis and treatment of many human conditions and in the development of new therapies. Our results also help elucidate the link between RBC dyscrasias and cardiovascular morbidity.

The tenet of high-affinity nitric oxide (NO) binding to a haemoglobin (Hb) has shaped our view of haem proteins and of small diffusible signaling molecules. Specifically, NO binds rapidly to haem iron in Hb (k approximately 10[7] M[-1] s[-1]) and once bound, the NO activity is largely irretrievable (Kd approximately 10[-5] s[-1]); the binding is purportedly so tight as to be unaffected by O2 or CO. However, these general principles do not consider the allosteric state of Hb or the nature of the allosteric effector, and they mostly derive from the functional behaviour of fully nitrosylated Hb, whereas Hb is only partially nitrosylated in vivo. Here we show that oxygen drives the conversion of nitrosylhaemoglobin in the 'tense' T (or partially nitrosylated, deoxy) structure to S-nitrosohaemoglobin in the 'relaxed' R (or ligand-bound, oxy) structure. In the absence of oxygen, nitroxyl anion (NO-) is liberated in a reaction producing methaemoglobin. The yields of both S-nitrosohaemoglobin and methaemoglobin are dependent on the NO/Hb ratio. These newly discovered reactions elucidate mechanisms underlying NO function in the respiratory cycle, and provide insight into the aetiology of S-nitrosothiols, methaemoglobin and its related valency hybrids. Mechanistic reexamination of NO interactions with other haem proteins containing allosteric-site thiols may be warranted.

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The oxidation of nitric oxide (NO) to nitrate by oxyhemoglobin is a fundamental reaction that shapes our understanding of NO biology. This reaction is considered to be the major pathway for NO elimination from the body; it is the basis for a prevalent NO assay; it is a critical feature in the modeling of NO diffusion in the circulatory system; and it informs a variety of therapeutic applications, including NOinhalation therapy and blood substitute design. Here we show that, under physiological conditions, this reaction is of little significance. Instead, NO preferentially binds to the minor population of the hemoglobin's vacant hemes in a cooperative manner, nitrosylates hemoglobin thiols, or reacts with liberated superoxide in solution. In the red blood cell, superoxide dismutase eliminates superoxide, increasing the yield of Snitrosohemoglobin and nitrosylated hemes. Hemoglobin thus serves to regulate the chemistry of NO and maintain it in a bioactive state. These results represent a reversal of the conventional view of hemoglobin in NO biology and motivate a reconsideration of fundamental issues in NO biochemistry and therapy.The chemistry of nitric oxide (NO) interactions with Hb has served as a ubiquitous model within the field of NO biochemistry. For example, the oxidative interaction of NO with oxyhemoglobin (oxyHb) to produce nitrate is considered to be the major route of NO catabolism (1-3) as well as a reliable method for assaying NO (4); likewise the unique ability of NO to induce displacement of a trans-imidazole heme ligand has been proposed as key to its activation of guanylyl cyclase (5). In the specific realm of the cardiovascular system, these reactions: are fundamental elements of models for NO diffusion (6, 7); played a crucial role in the identification of endothelium derived relaxing factor (6-9); and inform a variety of therapeutic applications, including NO-inhalation therapy (10, 11) and blood substitute design (12,13).Measurements of the rates of these reactions show that the NO-mediated oxidation of oxyHb to methemoglobin (metHb) is kinetically competitive with the binding of NO to unoccupied hemes in Hb-with specific rate constants of 3.7 ϫ 10 ) (16). Such a rapid route of NO metabolism is, however, difficult to reconcile with mammalian NO production rates (17), which are orders of magnitude too low to sustain physiological NO levels (10 nM-1 M) (7,(18)(19)(20), were NO to be freely consumed in these reactions. Previous studies of the NO oxyHb reaction, however, had been performed with NO concentrations 10-fold greater than protein (16). Under physiological conditions, the concentration ratio is starkly different, with NO concentrations 1,000-fold lower than Hb (20). Moreover, there is always a population of heme sites that are unoccupied. In highly oxygenated Hb, as found in arterial blood, this population is small (Ϸ1%) but is nevertheless in excess of NO. The influence of these vacant hemes, in the physiological situation, cannot be ignored; they might successfully compete f...

It is proposed that the bond between nitric oxide (NO) and the Hb thiol Cys-␤ 93 (SNOHb) is favored when hemoglobin (Hb) is in the relaxed (R, oxygenated) conformation, and that deoxygenation to tense (T) state destabilizes the SNOHb bond, allowing transfer of NO from Hb to form other (vasoactive) S-nitrosothiols (SNOs). However, it has not previously been possible to measure SNOHb without extensive Hb preparation, altering its allostery and SNO distribution. Here, we have validated an assay for SNOHb that uses carbon monoxide (CO) and cuprous chloride (CuCl)-saturated Cys. This assay is specific for SNOs and sensitive to 2-5 pmol. Uniquely, it measures the total SNO content of unmodified erythrocytes (RBCs) (SNORBC), preserving Hb allostery. In room air, the ratio of SNORBC to Hb in intact RBCs is stable over time, but there is a logarithmic loss of SNORBC with oxyHb desaturation (slope, 0.043). This decay is accelerated by extraerythrocytic thiol (slope, 0.089; P < 0.001). SNORBC stability is uncoupled from O2 tension when Hb is locked in the R state by CO pretreatment. Also, SNORBC is increased Ϸ20-fold in human septic shock (P ‫؍‬ 0.002) and the O2-dependent vasoactivity of RBCs is affected profoundly by SNO content in a murine lung bioassay. These data demonstrate that SNO content and O2 saturation are tightly coupled in intact RBCs and that this coupling is likely to be of pathophysiological significance.sepsis ͉ nitric oxide ͉ vascular physiology E vidence has accumulated for an S-nitrosothiol (SNO)-based vascular signaling system in which hemoglobin (Hb) reactions with nitric oxide (NO) transduce redox gradients into bioactivities (1-6). There is agreement that human Hb undergoes Snitrosylation at Cys-␤ 93 (3,(7)(8)(9)(10)(11). Erythrocytes are proposed to couple O 2 tension to the distribution of NO activities (such as control of blood flow) by linking the allosteric transition of Hb (12, 13) to conformation-dependent changes in the redox activity of this Cys-␤ 93 (13-18) and the stereochemistry of this SNO bond at Cys-␤ 93 (6, 7). Indeed, Cys-␤ 93 SNO in human Hb (SNOHb) can be crystallized only with the Hb tetramer in the relaxed (R, oxygenated) conformation; the SNO bond is unstable with Hb in the tense (T, deoxygenated) conformation (7). These observations support a paradigm in which NO binding to Cys-␤ 93 is favored in the R state and NO binding to Fe(II) (and͞or transnitrosation to an alternate thiol) is favored in the T state (19-21). Thus, the change in stability of Cys-␤ 93 SNO during Hb transition between R and T states may serve to couple regional O 2 gradients to the deployment or quenching of NO bioactivities in the microcirculation (2, 6, 22).However, assaying SNOHb has been problematic. First, detection of the SNO bond has required dilution and͞or pretreatment of Hb to (i) control for artifactual identification of nitrite and Fenitrosyl species and (ii) prevent autocapture of NO on Fe during analysis (8,19,(23)(24)(25). As a result, attempts to quantify Cys-␤ 93 SNO density can be biased ...

The ability of protein tyrosine kinases to phosphorylate a synthetic peptide was inhibited 51% by peroxynitritemediated nitration of tyrosine. Exposure of endothelial cells to peroxynitrite decreased the intensity of tyrosine phosphorylated proteins and increased the intensity of nitrotyrosine-containing proteins. Peroxynitrite-modified BSA was degraded by human red blood cell lysates. However, human plasma in a concentration-, time-, and temperature-dependent manner, removed the protein nitrotyrosine epitope. These results suggest that tyrosine nitration interferes with phosphorylation and targets proteins for degradation. Specific enzymatic process(es) for removing nitrotyrosine may be present in vivo.

There is substantial evidence that protein S-nitrosylation provides a significant route through which nitric oxide (NO)-derived bioactivity is conveyed. However, most examples of S-nitrosylation have been characterized on the basis of analysis in vitro, and relatively little progress has been made in assessing the participant forms of nitric-oxide synthase (NOS) or the dynamics of protein S-nitrosylation in situ. Here we utilize antibodies specific for the nitrosothiol (SNO) moiety to provide an immunohistochemical demonstration that protein Snitrosylation is coupled to the activity of each of the major forms of NOS. In cultured endothelial cells, SNOprotein immunoreactivity increases in response to Ca 2؉ -stimulated endothelial NOS (eNOS) activity, and in aortic rings, endothelium-derived and eNOS-mediated relaxation (EDRF) is coupled to increased protein Snitrosylation in both endothelial and associated smooth muscle cells. In cultured macrophages, SNO-protein levels increase upon cytokine induction of induced NOS (iNOS), and in PC12 cells, increased protein S-nitrosylation is linked to nerve growth factor induction of neuronal NOS (nNOS). In addition, we describe developmental and pathophysiological increases in SNOprotein immunoreactivity within human lung. These results, which demonstrate Ca 2؉ , neurohumoral, growth factor, cytokine, and developmental regulation of protein S-nitrosylation that is coupled to NOS expression and activity, provide unique evidence for the proposition that this ubiquitous NO-derived post-translational protein modification serves as a major effector of NOrelated bioactivity.Nitric oxide (NO), 1 generated by cell type-specific NO synthases (NOSs), has classically been characterized as a freely diffusible intercellular messenger that functions in target cells to subserve NOS-dependent signaling, which includes generation of endothelium-derived relaxing factor (EDRF) via eNOS, synaptic transmission and plasticity via nNOS, and antimicrobial activity via iNOS (see Ref. 1 for review). More recently, it has been proposed that S-nitrosylation of cysteine thiols may constitute a major route of NO trafficking through which NOrelated bioactivity is effected, serving as a ubiquitous posttranslational modification that regulates dynamically a broad functional spectrum of proteins (2-4). However, the analysis of protein S-nitrosylation in situ, originating with endogenous NOS activity, has been impeded by substantial technical barriers, and there is little direct evidence for cellular protein S-nitrosylation that can be ascribed specifically to the activity of any NOS isoform (4 -7). Here we show that S-nitrosylated proteins can be identified, on membrane blots and with immunohistochemistry, by antibodies that recognize the SNO moiety. We exploit this capacity to demonstrate ongoing and physiologically regulated protein S-nitrosylation that is coupled to the activity of each of the major forms of NOS in NO-generating cells and (in the case of endothelial NOS) their functional cellular partner...