Conversion of S-phenylsulfonylcysteine residues to mixed disulfides at pH 4.0: utility in protein thiol blocking and in protein-S-nitrosothiol detection.

A three step protocol for protein S-nitrosothiol conversion to fluorescent mixed disulfides with purified proteins, referred to as the thiosulfonate switch, is explored which involves: (1) thiol blocking at pH 4.0 using S-phenylsulfonylcysteine (SPSC); (2) trapping of protein S-nitrosothiols as their S-phenylsulfonylcysteines employing sodium benzenesulfinate; and (3) tagging the protein thiosulfonate with a fluorescent rhodamine based probe bearing a reactive thiol (Rhod-SH), which forms a mixed disulfide between the probe and the formerly S-nitrosated cysteine residue. S-Nitrosated bovine serum albumin and the S-nitrosated C-terminally truncated form of AdhR-SH (alcohol dehydrogenase regulator) designated as AdhR*-SNO were selectively labelled by the thiosulfonate switch both individually and in protein mixtures containing free thiols. This protocol features the facile reaction of thiols with S-phenylsulfonylcysteines forming mixed disulfides at mild acidic pH (pH = 4.0) in both the initial blocking step as well as in the conversion of protein-S-sulfonylcysteines to form stable fluorescent disulfides. Labelling was monitored by TOF-MS and gel electrophoresis. Proteolysis and peptide analysis of the resulting digest identified the cysteine residues containing mixed disulfides bearing the fluorescent probe, Rhod-SH.

Functional coupling of oxygen binding and vasoactivity in S-nitrosohemoglobin.

S-Nitrosohemoglobin (SNO-Hb) is a vasodilator whose activity is allosterically modulated by oxygen ("thermodyamic linkage"). Blood vessel contractions are favored in the oxygenated structure, and vasorelaxant activity is "linked" to deoxygenation, as illustrated herein. We further show that transnitrosation reactions between SNO-Hb and ambient thiols transduce the NO-related bioactivity, whereas NO itself is inactive. One remaining problem is that the amounts of SNO-Hb present in vivo are so large as to be incompatible with life were all the S-nitrosothiols transformed into bioactive equivalents during each arterial-venous cycle. Experiments were therefore undertaken to address how SNO-Hb conserves its NO-related activity. Our studies show that 1) increased O(2) affinity of SNO-Hb (which otherwise retains allosteric responsivity) restricts the hypoxia-induced allosteric transition that exchanges NO groups with ambient thiols for vasorelaxation; 2) some NO groups released from Cys(beta93) upon transition to T structure are autocaptured by the hemes, even in the presence of glutathione; and 3) an O(2)-dependent equilibrium between SNO-Hb and iron nitrosylhemoglobin acts to conserve NO. Thus, by sequestering a significant fraction of NO liberated upon transition to T structure, Hb can conserve NO groups that would otherwise be released in an untimely or deleterious manner.

Structural changes induced in p21Ras upon GAP-334 complexation as probed by ESEEM spectroscopy and molecular-dynamics simulation.

BACKGROUND: The means by which the protein GAP accelerates GTP hydrolysis, and thereby downregulates growth signaling by p21Ras, is of considerable interest, particularly inasmuch as p21 mutants are implicated in a number of human cancers. A GAP "arginine finger," identified by X-ray crystallography, has been suggested as playing the principal role in the GTP hydrolysis. Mutagenesis studies, however, have shown that the arginine can only partially account for the 10(5)-fold increase in the GAP-accelerated GTPase rate of p21. RESULTS: We report electron spin-echo envelope modulation (ESEEM) studies of GAP-334 complexed with GMPPNP bound p21 in frozen solution, together with molecular-dynamics simulations. Our results indicate that, in solution, the association of GAP-334 with GTP bound p21 induces a conformational change near the metal ion active site of p21. This change significantly reduces the distances from the amide groups of p21 glycine residues 60 and 13 to the divalent metal ion. CONCLUSIONS: The movement of glycine residues 60 and 13 upon the binding of GAP-334 in solution provides a physical basis to interpret prior mutagenesis studies, which indicated that Gly-60 and Gly-13 of p21 play important roles in the GAP-dependent GTPase reaction. Gly-60 and Gly-13 may play direct catalytic roles and stabilize the attacking water molecule and beta,gamma-bridging oxygen, respectively, in p21. The amide proton of Gly-60 could also play an indirect role in catalysis by supplying a crucial hydrogen bonding interaction that stabilizes loop L4 and therefore the position of other important catalytic residues.

The oxyhemoglobin reaction of nitric oxide.

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 NO-inhalation 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 S-nitrosohemoglobin 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.

Nitroxide side-chain dynamics in a spin-labeled helix-forming peptide revealed by high-frequency (139.5-GHz) EPR spectroscopy.

High-frequency electron paramagnetic resonance (EPR) spectroscopy has been performed on a nitroxide spin-labeled peptide in fluid aqueous solution. The peptide, which follows the single letter sequence, was reacted with the methanethiosulfonate spin label at the cysteine sulfur. The spin sensitivity of high-frequency EPR is excellent with less than 20 pmol of sample required to obtain spectra with good signal-to-noise ratios. Simulation of the temperature-dependent spectral lineshapes reveals the existence of local anisotropic motion about the nitroxide N-O bond with a motional anisotropy tau( perpendicular)/tau( parallel) ( identical with N) approaching 2.6 at 306 K. Comparison with previous work on rigidly labeled peptides suggests that the spin label is reorienting about its side-chain tether. This study demonstrates the feasibility of performing 140-GHz EPR on biological samples in fluid aqueous solution.

The effects of cryoprotection on the structure and activity of p21 ras: implications for electron spin-echo envelope modulation spectroscopy.

Electron spin-echo envelope modulation (ESEEM) spectroscopy is widely used to investigate the active sites of biological molecules in frozen solutions. Various cryoprotection techniques, particularly the addition of co-solvents, are commonly employed in the preparation of such samples. In conjunction with ESEEM studies of Mn(II) guanosine nucleotide complexes of p21 ras, we have investigated the effects of cryoprotection on the spectroscopy, the structure, and the activity of this protein. Echo decay times, which typically govern ESEEM spectral resolution, were found to vary linearly with the concentration of glycerol or methyl alpha-D-glucopyranoside (MG), with both additives equally effective on a per-mole basis. The effect of glycerol and MG on the ESEEM amplitudes of various protein nucleiwas studied in ras p21.Mn(II). 5'guanylylimido-diphosphate(p21.Mn(II)-GMPPNP) complexes: these additives did not alter the distances of these nuclei from the Mn(II) ion. In particular, in p21 incorporating [2H-3]Thr, the Mn(II)-[2H-3]Thr35 distance was found to be unaffected by the concentration of cryoprotectant or the rate of freezing. The proximity of the cryoprotectants to the Mn(II) ion was probed by 2H ESEEM in solutions made with d5-glycerol and d7-methyl alpha-D-glucopyranoside (d7-MG). In p21.Mn(II)GMPPNP, the large deuterium modulations from the d5-glycerol exhibit saturation behavior with increasing d5-glycerol concentration, implying that glycerol, a widely used cryoprotectant, replaces the aquo ligands of the Mn(II) ion. The interaction between the Mn(II) ion of p21 and MG, however, is less intimate: the deuterium ESEEM amplitudes are much smaller for samples prepared with d7-MG than with d5-glycerol. Several polyhydroxylic compounds were found to have essentially no effect on the ability of the guanosine 5'-triphosphate (GTP) hydrolysis activating protein, GAP334, to catalyze hydrolysis of p21. guanosine 5'-triphosphate. This observation implies that the introduction of cryoprotectant does not significantly perturb the structure of p21 and gives insight into the mechanism of the GTPase reaction.

The frozen solution structure of p21 ras determined by ESEEM spectroscopy reveals weak coordination of Thr35 to the active site metal ion.

BACKGROUND: The G protein p21 ras is a molecular switch in the signal transduction pathway for cellular growth and differentiation. Hydrolysis of tightly bound GTP alters the conformation of p21, terminating the signal. The coordination of the p21 residue Thr35 to Mg2+ in its active site, which has been observed in the crystal structure of p21 in complex with a GTP-analog GMPPNP but not with GDP, has been proposed to drive the conformational change accompanying nucleotide substitution and may have a role in the GTP hydrolysis reaction itself. However, previous electron spin-echo envelope modulation (ESEEM) studies of selectively 2H beta-threonine and 15N-threonine labeled p21.Mn2+ GMPPNP suggest that Thr35 only weakly coordinates the metal ion in the growth-active GTP-bound state of p21. RESULTS: A 13C beta-Thr35 to Mn2+ distance of 4.3 +/- 0.2 A and a 15N epsilon-Lys16 to Mn2+ distance of 5.3 +/- 0.2 A were determined from ESEEM spectra of the selectively 13C beta-Thr and 15N epsilon-Lys labeled p21.Mn2+ GMPPNP frozen solution structure. The 13C beta-Thr35 to Mn2+ distance is greater than that (3.16 A) observed in the crystal structure. In contrast, the 15N epsilon-Lys16 to Mn2+ distance is in good agreement with the 5.1 A crystal structure distance. CONCLUSIONS: The 13C beta of Thr35 is more distant from the active site Mn2+ in the frozen solution structure than in the crystal structure of p21.Mg2+ GMPPNP, indicating that Thr35 only weakly coordinates the metal ion in frozen solution. Thr35 coordination of the metal ion is therefore unlikely to drive the conformational change between GTP- and GDP-bound states of p21. Thr35 may be essential for GTPase-activating protein (GAP)-stimulated GTP hydrolysis and/or signal transduction for other reasons.

High frequency (139.5 GHz) electron paramagnetic resonance characterization of Mn(II)-H2(17)O interactions in GDP and GTP forms of p21 ras.

As a molecular switch, the ras protein p21 undergoes structural changes that couple recognition sites on the protein surface to the guanine nucleotide-divalent metal ion binding site. X-ray crystallographic studies of p21 suggest that coordination between threonine-35 and the divalent metal ion plays an important role in these conformational changes. Recent ESEEM studies of p21 in solution, however, place threonine-35 more distant from the metal and were interpreted as weak or indirect coordination of this residue. We report high frequency (139.5 GHz) EPR spectroscopy of p21.Mn(II) complexes of two guanine nucleotides that probes the link between threonine-35 and the divalent metal ion. By analysis of high-frequency EPR spectra, we determine the number of water molecules in the first coordination sphere of the manganous ion to be four in p21.Mn(II).GDP, consistent with prior low-frequency EPR and X-ray crystallographic studies. In the complex of p21 with a GTP analog, p21.Mn(II).GMPPNP, we determine the hydration number to be 2, also consistent with crystal structures. This result rules out indirect coordination of threonine-35 in the solution structure of p21.Mn(II).GMPPNP, and implicates direct, weak coordination of this residue as suggested by Halkides et al. [(1994) Biochemistry 33,4019]. The 17O hyperfine coupling constant of H2(17)O is determined as 0.25 mT in the GDP from and 0.28 mT in the GTP form. These values are similar to reported values for 17O-enriched aquo ligands and some phosphato ligands in Mn(II) complexes. The high magnetic field strength (4.9 T) employed in these 139.5 GHz EPR measurements leads to a narrowing of the Mn(II) EPR lines that facilitates the determination of 17O hyperfine interactions.

High frequency (139.5 GHz) electron paramagnetic resonance spectroscopy of the GTP form of p21 ras with selective 17O labeling of threonine.

Electron paramagnetic resonance spectroscopy at 139.5 GHz has been used to study p21 ras complexed with Mn(II) and guanosine 5'-(beta, gamma-imidotriphosphate), an analog of GTP. The p21 sample studied was selectively labeled with [17O gamma]threonine to a final enrichment of 30%. A Mn(II)-17O hyperfine interaction was observed, but the value of the coupling constant, 0.11 +/- 0.04 mT, is the smallest such value yet reported. Ab initio calculations indicate that this value is consistent with direct coordination of the threonine hydroxyl group and provide an estimate for the Mn(II)-17O bond length of 2.7 A. The measured hyperfine coupling constant and associated bond length starkly contrast with typical values for Mn(II)-17O coordination complexes, namely, approximately 0.25 mT and approximately 2.2 A, respectively. This contrast underscores the peculiar weakness of this Mn(II)-O interaction in p21 and persuasively argues that the nucleotide-induced conformational change, which is known to encompass the region of p21 involving Thr35, is not driven by Mn(II) coordination of the Thr35 hydroxyl group.

Polynitrosylated proteins: characterization, bioactivity, and functional consequences.

Chemical modification of proteins is a common theme in their regulation. Nitrosylation of protein sulfhydryl groups has been shown to confer nitric oxide (NO)-like biological activities and to regulate protein functions. Several other nucleophilic side chains -- including those with hydroxyls, amines, and aromatic carbons -- are also potentially susceptible to nitrosative attack. Therefore, we examined the reactivity and functional consequences of nitros(yl)ation at a variety of nucleophilic centers in biological molecules. Chemical analysis and spectroscopic studies show that nitrosation reactions are sustained at sulfur, oxygen, nitrogen, and aromatic carbon centers, with thiols being the most reactive functionality. The exemplary protein, BSA, in the presence of a 1-, 20-, 100-, or 200-fold excess of nitrosating equivalents removes 0.6 +/- 0.2, 3.2 +/- 0.4, 18 +/- 4, and 38 +/- 10, respectively, moles of NO equivalents per mole of BSA from the reaction medium; spectroscopic evidence shows the proportionate formation of a polynitrosylated protein. Analogous reaction of tissue-type plasminogen activator yields comparable NO protein stoichiometries. Disruption of protein tertiary structure by reduction results in the preferential nitrosylation of up to 20 thus-exposed thiol groups. The polynitrosylated proteins exhibit antiplatelet and vasodilator activity that increases with the degree of nitrosation, but S-nitroso derivatives show the greatest NO-related bioactivity. Studies on enzymatic activity of tissue-type plasminogen activator show that polynitrosylation may lead to attenuated function. Moreover, the reactivity of tyrosine residues in proteins raises the possibility that NO could disrupt processes regulated by phosphorylation. Polynitrosylated proteins were found in reaction mixtures containing interferon-gamma/lipopolysaccharide-stimulated macrophages and in tracheal secretions of subjects treated with NO gas, thus suggesting their physiological relevance. In conclusion, multiple sites on proteins are susceptible to attack by nitrogen oxides. Thiol groups are preferentially modified, supporting the notion that S-nitrosylation can serve to regulate protein function. Nitrosation reactions sustained at additional nucleophilic centers may have (patho)physiological significance and suggest a facile route by which abundant NO bioactivity can be delivered to a biological system, with specificity dictated by protein substrate.

Characterization of the active site of p21 ras by electron spin-echo envelope modulation spectroscopy with selective labeling: comparisons between GDP and GTP forms.

Selectively labeled samples of human H- or N-ras p21 ligated to MnIIGDP or MnIIGMPPNP were studied by electron spin-echo envelope modulation spectroscopy in order to define the protein environment around the divalent metal. We incorporated [4-13C]-labeled Asx into p21.MnIIGDP and found that the distance from the carboxyl 13C of Asp57 to MnII is approximately 4.1 A. Our result is consistent with indirect coordination of this residue to the metal. From a [2-2H]Thr-labeled sample, we estimate that the distance from the MnII ion to the 2H of Thr35 is at least 5.8 A. Thus, the only protein or nucleotide ligands to the metal appear to be Ser17 and the beta-phosphate of GDP, as previously reported [Larsen, R. G., Halkides, C. J., Redfield, A. G., & Singel, D. J. (1992b) J. Am. Chem. Soc. 114, 9608-9611]. In the 5'-guanylylimido diphosphate (GMPPNP) form of p21, Thr35 has been reported by X-ray crystallography to be a ligand of the metal via its hydroxyl group, and this residue appears to play a key role in the biologically important conformational change upon nucleotide substitution [Pai, E. F., Krengel, U., Petsko, G., Goody, R. S., Kabsch, W., & Wittinghofer, A. (1990) EMBO J. 9, 2351-2359]. The ESEEM spectrum of p21.MnIIGMPPNP labeled with [2-2H]Thr yields a MnII-2H distance of 4.9 A, a distance inconsistent with strong coordination. A sample of p21 in which the Thr residues were fully labeled with 13C and 15N yielded a value of 5.0 A for the distance from MnII to the amide nitrogen of Thr35, while the 13C signal is much smaller than expected if Thr35 were coordinated. A [15N]serine/glycine-labeled sample gives a distance to the amide 15N of Ser17 of 3.9 A, consistent with the X-ray structure; a [4-13C]-labeled Asx sample of p21 gives a distance of approximately 4 A between MnII and the label of Asp57, again implying indirect coordination. Both of these values are very similar to those found for the GDP form of the protein. The results for Thr35, however, reveal a structural difference between the GDP and GTP forms in the region of Thr35. In addition, the position of this residue is found to be different from the crystal structure and in a manner suggesting that the metal ligation of Thr35 does not drive the conformational change that accompanies nucleotide substitution.

Nitric oxide in the central nervous system.

1. The reactions of nitric oxide with superoxide can lead to neurotoxicity through formation of peroxynitrite, and not by NO. alone, at least under our conditions. 2. Transfer of NO+ groups to thiol(s) on the NMDA receptor can lead to neuroprotection by inhibiting Ca2+ influx. These findings suggest that cell function can be controlled by, or through, protein S-nitrosylation, and raise the possibility that the NO group may initiate signal transduction in or at the plasma membrane. 3. The local redox milieu of a biological system is of critical importance in understanding NO actions as disparate chemical pathways involving distinct redox related congeners of NO may trigger neurotoxic or neuroprotective pathways. These claims are highlighted in the CNS by the recent finding that tissue concentrations of cysteine approach 700 microM in settings of cerebral ischemia (Slivka and Cohen, 1993); these levels of thiol would be expected to influence the redox state of the NO group. 4. Finally, our findings suggest novel therapeutic strategies. For example, downregulation of NMDA receptor activity via S-nitrosylation with NO+ donors could be implemented in the treatment of focal ischemia, AIDS dementia, and other neurological disorders associated, at least in part, with excessive activation of NMDA receptors.

Peroxynitrite-induced rat colitis--a new model of colonic inflammation.

BACKGROUND: Excessive production of nitric oxide, characteristic of inflamed states, may have deleterious effects through its facile conversion (in the presence of O2) to peroxynitrite, which promotes lipid and sulfhydryl oxidation. This study assessed the effect of peroxynitrite on the rat colon. METHODS: Peroxynitrite was administered intrarectally to rats. One, 3, 7, and 21 days after treatment, a distal colonic segment was isolated and tissue was obtained for histological evaluation and determination of myeloperoxidase activity and NOX, and eicosanoids generation. RESULTS: Within 24 hours, the exposed segment was edematous and congested with occasional hemorrhagic mucosal ulceration. On day 7, the lumen was narrow; at day 21, there were signs of stenosis. Histological analysis showed transmucosal necrosis, acute inflammation, and exudative edema 24 hours after treatment. Surface re-epithelization and infiltration of granulation tissue were present at 1 week. Resolution of edema, mucin repletion, thickening of muscularis mucosa and propria, and fibrosis were observed at 3 weeks. Significant increase in NOX generation and myeloperoxidase and NO synthase activities were observed at 24 hours, whereas enhanced leukotriene generation was observed only at 21 days. CONCLUSIONS: Peroxynitrite-induced colonic inflammation provides a novel model of NO-related tissue injury and offers the opportunity to further explore the potential role of NO in the pathogenesis of inflammatory bowel disease.

Endogenous nitrogen oxides and bronchodilator S-nitrosothiols in human airways.

Recent discoveries suggesting essential bioactivities of nitric oxide (NO.) in the lung are difficult to reconcile with the established pulmonary cytotoxicity of this common air pollutant. These conflicting observations suggest that metabolic intermediaries may exist in the lung to modulate the bioactivity and toxicity of NO.. We report that S-nitrosothiols (RS-NO), predominantly the adduct with glutathione, are present at nano- to micromolar concentrations in the airways of normal subjects and that their levels vary in different human pathophysiologic states. These endogenous RS-NO are long-lived, potent relaxants of human airways under physiological O2 concentrations. Moreover, RS-NO form in high concentrations upon administration of NO. gas. Nitrite (10-20 microM) is found in airway lining fluid in concentrations linearly proportional to leukocyte counts, suggestive of local NO. metabolism. NO. itself was not detected either free in solution or in complexes with transition metals. These observations may provide insight into the means by which NO. is packaged in biological systems to preserve its bioactivity and limit its potential O2-dependent toxicity and suggest an important role for NO. in regulation of airway luminal homeostasis.

A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds.

Congeners of nitrogen monoxide (NO) are neuroprotective and neurodestructive. To address this apparent paradox, we considered the effects on neurons of compounds characterized by alternative redox states of NO: nitric oxide (NO.) and nitrosonium ion (NO+). Nitric oxide, generated from NO. donors or synthesized endogenously after NMDA (N-methyl-D-aspartate) receptor activation, can lead to neurotoxicity. Here, we report that NO.- mediated neurotoxicity is engendered, at least in part, by reaction with superoxide anion (O2.-), apparently leading to formation of peroxynitrite (ONOO-), and not by NO. alone. In contrast, the neuroprotective effects of NO result from downregulation of NMDA-receptor activity by reaction with thiol group(s) of the receptor's redox modulatory site. This reaction is not mediated by NO. itself, but occurs under conditions supporting S-nitrosylation of NMDA receptor thiol (reaction or transfer of NO+). Moreover, the redox versatility of NO allows for its interconversion from neuroprotective to neurotoxic species by a change in the ambient redox milieu. The details of this complex redox chemistry of NO may provide a mechanism for harnessing neuroprotective effects and avoiding neurotoxicity in the central nervous system.

Antiplatelet properties of protein S-nitrosothiols derived from nitric oxide and endothelium-derived relaxing factor.

S-nitrosothiols may serve as carriers in the mechanism of action of endothelium-derived relaxing factor (EDRF) by stabilizing the labile nitric oxide (NO) radical from inactivation by reactive species in the physiological milieu and by delivering NO to the heme activator site of guanylyl cyclase. Low-molecular-weight thiols, such as cysteine and glutathione, form S-nitrosothiol adducts with vasodilatory and antiplatelet properties, and protein thiols can interact in the presence of NO and/or EDRF to form uniquely stable S-nitroso-proteins. We now show that the S-nitroso-proteins, S-nitroso-albumin, S-nitroso-tissue type plasminogen activator, and S-nitroso-cathepsin B, have potent antiplatelet effects with an IC50 of approximately 1.5 microM. In the dog, S-nitroso-albumin inhibits ex vivo platelet aggregation and significantly prolongs the template bleeding time from 2.15 +/- 0.13 (mean +/- SEM) to 9.70 +/- 1.24 minutes. The antiplatelet action of S-nitroso-proteins is associated with the stimulation of guanylyl cyclase and a significant decrease in fibrinogen binding to platelets. S-Nitroso-proteins undergo thiol-nitrosothiol exchange with low-molecular-weight thiols to form low-molecular-weight S-nitroso-thiols, and they also interact directly with the platelet surface, both of which processes facilitate generation of NO. These data suggest that S-nitroso-proteins are potent antiplatelet agents and may be intermediates in the antiplatelet mechanism of EDRF action.

Biochemistry of nitric oxide and its redox-activated forms.

Nitric oxide (NO.), a potentially toxic molecule, has been implicated in a wide range of biological functions. Details of its biochemistry, however, remain poorly understood. The broader chemistry of nitrogen monoxide (NO) involves a redox array of species with distinctive properties and reactivities: NO+ (nitrosonium), NO., and NO- (nitroxyl anion). The integration of this chemistry with current perspectives of NO biology illuminates many aspects of NO biochemistry, including the enzymatic mechanism of synthesis, the mode of transport and targeting in biological systems, the means by which its toxicity is mitigated, and the function-regulating interaction with target proteins.

S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds.

Endothelium-derived relaxing factor (EDRF) activity has been attributed to the highly labile nitric oxide radical (NO). In view of the fact that the plasma and cellular milieux contain reactive species that can rapidly inactivate NO, it has been postulated that NO is stabilized by a carrier molecule that preserves its biological activity. Reduced thiol species are candidates for this role, reacting readily in the presence of NO to yield biologically active S-nitrosothiols that are more stable than NO itself. Because sulfhydryl groups in proteins represent an abundant source of reduced thiol in biologic systems, we examined the reaction of several sulfhydryl-containing proteins of diverse nature and function upon exposure to authentic NO and EDRF. We demonstrate that S-nitroso proteins form readily under physiologic conditions and possess EDRF-like effects of vasodilation and platelet inhibition. These observations suggest that S-nitrosothiol groups in proteins may serve as intermediates in the cellular metabolism of NO and raise the possibility of an additional type of cellular regulatory mechanism.

Electron nuclear double resonance spectroscopy of a hen egg-white lysozyme--Cu2+ complex in tetragonal single crystals.

We have used the electron paramagnetic resonance of Cu2+ bound in a tetragonal single crystal of hen egg-white lysozyme to obtain the electron nuclear double resonance spectra of protons in the vicinity of the Cu2+ at the site designated as B by Teichberg et al. [Teichberg, V. I., Sharon, N., Moult, J., Smilansky, A. & Yonath, A. (1974) J. Mol. Biol. 87, 357-368]. The values of the hyperfine interaction parameters and the coordinates of eight protons are reported. The configuration of the H2O molecules coordinated to the Cu2+ and their relationships to the protein molecule structure are discussed.