Thionitroxides, RSNHO^•:  The Structure of the SNO Moiety in “S-Nitrosohemoglobin”, A Possible NO Reservoir and Transporter

Abstract

Considerable evidence suggests that the S-nitrosation of cysteine residues, thought to regulate the activities of many proteins, is involved in the uptake and intracellular trafficking of NO. 1 Indeed, to absorb the neutral NO with thiols, a redox reaction is needed to form Snitrosothiols (RSNOs). [1][2][3] A structure of S-nitrosocysteine in hemoglobin proteins was first reported by Arnone and coworkers in 1998. 4a Interestingly, the resulting protein structure obtained under nonbiological conditions (NO pressure, anaerobic) exhibits unexpected C-S-N-O dihedral angles of 88° and -76° for Cys93 of β1 and β2 subunits. The crystallographic results contradict the planar geometries of all RSNOs in small molecule crystallographic data, 5 and from quantum mechanical calculations. 6 Recently, Arnone et al. refined the X-ray data again and reported that the electron density of the SNO-Cys98(F9)β could not be fit with a planar syn or an anti S-nitrosothiol model. They found C-S-N-O dihedral angles between 70° and 90° for SNO-Cys93(F9)β1 and SNO-Cys93(F9)β2. (Fig. 1) 4b To explain this, they proposed that a N-centered radical, Cys-S-N·-OH, was present and stabilized by hydrogen bonding with Val98. Such a radical was previously proposed as an intermediate in reactions of NO with thiols. 7 However, the SNO moiety in the Cys93(F9)β2 subunit is unlikely to be stabilized in this way, because the shortest O---O distance between the oxygen atom of the studied SNO species and the nearest oxygen of Lys144 is about 3.84 Å, a distance too long for significant OH---O hydrogen bonding interactions.By contrast, Montfort and coworkers have reported that the nitric oxide transport protein, nitrophorin cNP, 8 couples reduction and oxidation of a heme moiety with reversible Snitrosation of a cysteine residue (PDB: 1Y21). This S-nitrosothiol species has a C-S-N-O dihedral angle of 0°, coinciding with a planar CSNO geometry in small molecule crystallographic data. The cysteine sulfide coordinating to ferric heme is the key to the Snitrosothiol formation in this protein. Unlike nitrophorins, the NO-bound cysteine in hemoglobin is 10 Å (Fe---S) from the closest heme.To determine the identity of the ~90° CSNO moiety in the purported S-nitrosohemoglobin, we have explored all four types of SNO species (RSNO, RS-N·-OH, RS-NH-O·, and RS-NH-OH) that could be candidates for the observed SNO species, using simple models (R = Me) that can be computed at a high level. By comparison of the calculated structures to the experimental crystallographic data, we have discovered that the thionitroxide radical (Cys-S-NH-O·) is consistent with the observed SNO structures in both β1 and β2 subunits.Correspondence to: K. N. Houk. Supporting Information Available: Complete reference 9, the NO-modified hemoglobin, and the detailed structures and energetics of stationary points on potential energy surfaces of the four SNO species. These materials are available free of charge via the Internet at http://pubs.acs.org. NIH Public AccessAuthor Manuscript J Am Chem Soc...

S-Nitrosylation-induced Conformational Change in Blackfin Tuna Myoglobin

“…Quantum mechanical calculations demonstrate that two energy minima exist for the C-S-N-O dihedral angle, at 0 and 180°, due to delocalization of the N ϭ O electrons over the S-N bond (a phenomenon that may be familiar from the planar peptide bond within the backbone of proteins). This results in a slightly shorter S-N bond and a 12-kcal/mol energy barrier for rotation of the dihedral between 0 and 180° (39). Both conformations of Cys-10 -SNO that we observed in myoglobin closely matched the predicted and observed geometric parameters for S-nitrosothiols.…”

An S -nitrosothiol (SNO) synthase function of hemoglobin that utilizes nitrite as a substrate

“…A clue to the importance of reactant concentrations on this chemistry emerged from EPR studies, which revealed, in the aftermath of mixing very high concentrations of nitrite and deoxyHb, essentially equal subunit populations of nitrosyl heme [␣Fe(II)NO Ϸ ␤Fe(II)NO], whereas spectra obtained under conditions that simulate key aspects of the in vivo situation exhibit substantial ␤Fe(II)NO preference (16). In addition, the importance of duration of reaction on product distribution was recognized: Aging of NO-deoxyHb samples that is incurred over the lengthy course of the nitrite reaction enables competing chemistry, including redistribution of NO from ␤-to ␣-chains, reductive loss of NO to HNO and quenching of radicals in Hb (4,7,(13)(14)(15)34). Because previous work has suggested collectively that the efficacy of transfer of NO groups from heme to ␤Cys-93 (to form a bioactive SNO) might require not only physiological NO͞Hb ratios and HbNO concentrations (Ͻ 1 M) but also preferential processing of NO within the ␤-chain (14-17), the conditions used in prior work were not optimized for the study of SNO formation.…”

Assessment of nitric oxide signals by triiodide chemiluminescence

“…The dynamic distribution of protein and low-molecular-weight NO compounds that subserve NO transport and signaling instantiate the variation in both FeNO and SNO reactivities (4,6,13,[16][17][18][19][20][21][22][23][24] (5,6,13,(16)(17)(18)(19)(20)(21)(22)25). Accordingly, bond dissociation energies of RSNO are reported to vary from Ϸ22 to 32 kcal⅐mol Ϫ1 (6, 26), and the dissociation constants of FeNO can vary by a factor of Ͼ10 6 (13, 23, 24), translating to intrinsic FeNO/SNO lifetimes ranging from seconds to years.…”

“…Accordingly, bond dissociation energies of RSNO are reported to vary from Ϸ22 to 32 kcal⅐mol Ϫ1 (6, 26), and the dissociation constants of FeNO can vary by a factor of Ͼ10 6 (13, 23, 24), translating to intrinsic FeNO/SNO lifetimes ranging from seconds to years. Environmental factors that have been reported to influence SNO stability and reactivity, directly or through elicited conformational changes in proteins, include pH (low and high) (5,6,20,26), metal ions (Ca, Mg, Cu, and Fe) (6,14,20,27,28), nucleophiles (ascorbate, thiolate, and amine) (6, 13), local hydrophobicity (denaturants) (29), oxidants and reductants (6, 19), proteolytic enzymes (30), alkylators (31), O 2 tension (5, 32), and various intramolecular interactions (H-bonding, S-, N-, O-coordination, and aromatic residue interactions) (6,16,20,22,(33)(34)(35)(36). Many of these factors also affect FeNO stability (17,23,24).…”

“…Additionally, NO can be transported in endocrine or paracrine fashion by reacting with heme iron and cysteine thiols in proteins [hemoglobin (Hb) and albumin] and peptides (glutathione and cysteinlyglycine) to form NO adducts with longer biological lifetimes (3-5); release of NO bioactivity from stable adducts is effected by allosteric and redox-based mechanisms that alter FeNO or S-nitrosothiol (SNO) reactivity (5, 6). An updated discussion of the factors influencing reactivity of S-nitrosohemoglobin, S-nitrosoalbumin, and low-molecular-weight SNO in the context of vasoregulation (5-15) can be found in supporting information (SI) Text.The dynamic distribution of protein and low-molecular-weight NO compounds that subserve NO transport and signaling instantiate the variation in both FeNO and SNO reactivities (4,6,13,[16][17][18][19][20][21][22][23][24] (5,6,13,(16)(17)(18)(19)(20)(21)(22)25). Accordingly, bond dissociation energies of RSNO are reported to vary from Ϸ22 to 32 kcal⅐mol Ϫ1 (6, 26), and the dissociation constants of FeNO can vary by a factor of Ͼ10 6 (13, 23, 24), translating to intrinsic FeNO/SNO lifetimes ranging from seconds to years.…”