Vibrational Signatures of S-Nitrosoglutathione as Gaseous, Protonated Species

Abstract

Gas-phase ions of protonated l-glutathione as native species, [GSH + H](+), and S-nitroso derivative, [GSNO + H](+), have been generated by electrospray ionization and probed via infrared multiple photon dissociation (IRMPD) action spectroscopy. Insight into the conformational landscape is gained from interpretation of the IR spectra aided by high-level theoretical calculations, which enables structural assignment disclosing both the site of protonation and the intramolecular hydrogen-bond network. Calculations yield the low-energy structures of [GSNO + H](+). A admixture of the four most stable ones (SN1, AN1, SN2, and AN2) is apt to account for the experimental IRMPD spectra obtained in both the 1000-2000 and the 3100-3700 cm(-1) spectral ranges. The most stable form of [GSNO + H](+), SN1, protonated at the amino group, presents a syn conformation at the S-N (partial) double bond and all peptidic carbonyls involved in (strong) C═O···H-N hydrogen bonds, so allowing closure of a C5 (β-strand), two C7 (γ-turn), and one C9-membered rings. An appreciable barrier to rotation of 43 kJ mol(-1) about the S-N bond is found to separate SN1 from the analogous anti isomer AN1, which lies only 0.70 kJ mol(-1) higher in free energy. Conformers obtained for [GSH + H](+) are very similar to the [GSNO + H](+) counterparts, indicating that the S-nitrosation motif does not affect significantly the geometry of the peptide. The observed ν(NO) signatures at 1622 and 1690 cm(-1), merged with other absorptions, are revealed by their sensitivity to (15)NO isotope labeling and by comparison with the IRMPD spectrum of native [GSH + H](+), providing a diagnostic probe for the S-nitrosation feature in natural peptides.

Amino Acid Oxidation: A Combined Study of Cysteine Oxo Forms by IRMPD Spectroscopy and Simulations

“…Refined relative enthalpies (or Gibbs free energies) of the [cysSO x ] − ( x =1, 2, 3) isomers and conformers were also obtained from single‐point energy calculations at the MP2/6‐311+G(d,p) level by using the B3LYP geometries, and combining the MP2 electronic energies with thermal corrections (298 K) from B3LYP calculations …”

“…[63] Refined relative enthalpies (or Gibbs free energies) of the [cysSO x ] À (x = 1, 2, 3) isomers and conformers were also obtained from single-point energy calculations at the MP2/6-311 + G(d,p) level by using the B3LYP geometries, and combining the MP2 electronic energies with thermal corrections (298 K) from B3LYP calculations. [27] Spectral assignment was achieved by comparing experimental IRMPD spectra to calculated IR spectra. Because the harmonic approximation was known to overestimate the computed frequencies, the harmonic frequencies were uniformly scaled by 0.967, which improved agreement between calculated and measured absorptions.…”

“…The unambiguous and detailed characterization of the structural and dynamic behavior of these transient intermediates in isolated environments, in which external interferences are absent, will help to reveal intrinsic properties relevant to their biological activity. The inherent bonding and conformational features of cysteine in its (de)protonated, nitrosated, and metal‐tagged forms have been previously interrogated both in the free amino acid and in the residue embodied in the glutathione peptide by a joint computational and experimental infrared multiple photon dissociation (IRMPD) assay. IRMPD spectroscopy, coupled with ESI tandem mass spectrometry (MS/MS), is a pivotal tool to provide direct structural clues about gaseous (bio)molecular ions, including (de)protonation site, metal‐binding patterns, and local intramolecular interactions in both native and modified amino acids and peptides, DNA/RNA bases and nucleotides, cluster ions, and fragmentation products …”

“…[18] Recently,avariety of mass spectrometric studies have been able to identify the reactive intermediates formed by (interfacial) ozonolysis of deprotonated cysteineatthe air-liquid interface, [5,19] to probe the stability and fragmentation behavior of intact and modified sulfinyl radical ions, [20,21] and elucidate the intrinsic mechanism involved in the formationo fc ysteine oxo forms [cysSO x ] À (x = 1, 2, 3) and sulfenate radicala nions in the gas phase. [22] The unambiguous and detailed characterization of the structurala nd dynamic behavior of theset ransient intermediates in isolated environments, in which external interferences are absent,w ill help to reveali ntrinsic properties rele-vant to their biological activity.T he inherentb onding and conformationalf eatureso fc ysteine in its (de)protonated, [23,24] nitrosated, [25] and metal-tagged [26] forms have been previously interrogated both in the free amino acid and in the residue embodied in the glutathione peptide [27] by aj oint computational and experimental infrared multiple photon dissociation (IRMPD) assay.I RMPD spectroscopy,c oupled with ESI tandem mass spectrometry (MS/MS), is ap ivotalt ool to provide direct structural clues about gaseous (bio)molecular ions, [28][29][30][31][32][33] including (de)protonations ite, metal-binding patterns,a nd local intramolecular interactions in both native and modified amino acids and peptides, [34][35][36][37][38][39][40][41][42] DNA/RNA bases and nucleotides, [43][44][45] cluster ions, [46] and fragmentation products. [47] We now examine the intrinsic chemical properties and geometric features of the conjugate bases of cysteine sulfenic, sulfinic, and sulfonica cids, [cysSO x ] À (x = 1, 2, 3), by meanso fE SI/ MS-IRMPD spectroscopy in the highly informativem id-IR spectral range (750-1900 cm À1 )i nc ombination with ab initio...…”

Electronic Properties and Ionic Photodissociation of Thionitrite Compounds RSNO [R=(CH3)3C- and (CH3)2CH-]

“…The characteristic ν(NO) stretching vibration is clearly in the 1460–1490 cm –1 range in the IRMPD spectrum of deprotonated species, providing a signature for the S‐nitrosation process . Most recently, ions of the S‐nitroso derivative of L‐glutathione were generated in the gas phase and the conformational properties determined by interpreting the infrared spectra …”