Reactions of glutathionylcobalamin with nitroxyl and its donor, Angeli’s salt

Abstract We investigated the reaction between glutathionylcobalamin (GSCbl), a tight complex of cobalamin with glutathione (GSH), and nitroxyl (HNO) produced upon decomposition of Angeli’s salt using ultraviolet-visible spectroscopy and 1H NMR. The reaction in neutral and alkaline media leads to formation of nitrosylcobalamin (NOCbl). Simulation of the kinetic traces using the ChemMech program package suggested that the mechanism of the process involves complexation between HNO and GSCbl and further decomposition of the complex into the products. The complex participates in an acid-base equilibrium (pKa = 8.9), and the deprotonated form decomposes to NOCbl more rapidly than the protonated species. At pH 7.4, the reaction of HNO with GSCbl proceeds ca.103-times more slowly than with free GSH. GSCbl can react directly with the monoprotonated anion of Angeli’s salt (HN2O3−) to produce nitrocobalamin (NO2Cbl). The reaction between GSCbl and HN2O3− is insignificant in an alkaline medium and is accelerated upon acidification of the medium. The critical step of the process is the complexation between GSCbl and HN2O3−. The produced complex is involved in an acid-base equilibrium (pKa = 8.1): the protonated form can be transformed to NO2Cbl and GSNHO−, whereas the deprotonated species decompose to initial reactants.


Introduction
Nitric oxide (NO) is a well-recognized signaling molecule that influences several physiological processes in living organisms.Its endogenous production occurs primarily via the oxidation of arginine [1].NO exhibits high reactivity toward numerous biological molecules, and the most important of its biological targets are metal centers [2], e.g.heme iron(II) is capable of rapidly binding NO [3].Reactions between NO and thiols, hydrogen sulfide, and alcohols proceed substantially less efficiently and produce nitroxyl (HNO) [4][5][6].Similar to NO, HNO mediates processes in the cardiovascular [7] and nervous systems [8].However, their reactivity toward several biological substrates is different; in particular, HNO is ca.10 5 -fold more reactive toward thiols [5,9].The values of the rate constant for the reaction between HNO and glutathione (GSH) vary, i.e. 2.0 � 10 6 [9], 3.1 � 10 6 [10], and 7.6 � 10 6 [11] M −1 s −1 at pH 7.4.The process has a relatively complex mechanism, and it is initiated by nucleophilic attack of thiol group to HNO to give GS-NHOH, which can be further converted to sulfinamide decomposing to sulfinic acid and ammonia, or it can react with a second GSH molecule to produce glutathione disulfide and hydroxylamine [12].
The inhibition of Cbl-dependent enzymes by NO, i.e. methionine synthase via Cbl(I) to Cbl(II) oxidation [34] and methylmalonyl-CoA-mutase via NO binding to Cbl(II)-intermediate [35], provides evidence for the reaction between cobalamins and NO in vivo.Moreover, (H 2 O)(HO − )Cbi is capable of NO scavenging in vitro via formation of NOCbi [36].Due to the substantial aerobic stability of nucleotide-free nitrosylcorrinoids [37,38], they might find application in medicine as hypotensive [39] and wound-healing agents [40].
Despite the absence of information on the influence of HNO on Cbl-dependent processes in vivo, it is anticipated due to the high reactivity of HNO to some corrinoid species.In particular, HNO generated upon decomposition of Angeli's salt [41] or Piloty's acid [42] is rapidly scavenged by H 2 OCbl to give NOCbl.Interestingly, this reaction can proceed with hydroxocobalamin (HOCbl) without its preliminary protonation to H 2 OCbl [43].Cbl(II) also reacts with HNO to produce NOCbl via a two-step process involving the formation of Cbl(I) [43].Moreover, H 2 OCbl can directly receive HNO from its donors (e.g.Angeli's salt [41], Piloty's acid [42], NONOates [44]); likewise, GSCbl reacts with diethylamine NONOate, producing NOCbl [45].Taking into account the rapid formation of nitrosylcobalamin in these reactions, which is resistant to intracellular processing by CblC protein to cob(II)alamin [33], a cofactor precursor form, HNO, may inhibit some processes in the intracellular metabolism of Cbls.The reaction between GSCbl and HNO remains unstudied; however, taking into account the high reactivity of HNO toward thiols [9-12] and the pronounced redox properties of GSCbl [20,46,47], it is anticipated.Moreover, reactions of HNO with thiolate ligands bound in metal complexes are poorly investigated as well, although they may contribute to its signaling.Thus, here we assessed the pathway of nitrosylcobalamin formation involving GSCbl (Figure 1(A)) and HNO generated upon decomposition of Angeli's salt (Figure 1(B)), which might inhibit intracellular processing of Cbls.

Instrumentation
UV-vis spectra were recorded with a thermostated (±0.1 � C) Cary 50 UV-vis and Shimadzu UV-1800 spectrophotometers in quartz cells under anaerobic conditions.The NMR measurements were conducted using a Bruker Avance III 500 NMR spectrometer in deoxygenated deuterium oxide (D 2 O) with a purity of 99.9% from Cambridge Isotope Laboratories Inc.The pH values of the solutions were determined by using a Multitest IPL-103 pH-meter (SEMICO) equipped with an ESK-10601/7 electrode (Izmeritelnaya tekhnika) filled with a 3.0 M KCl solution.The electrode was preliminarily calibrated using standard buffer solutions (pH 1.65-12.45).

Data analysis
Experimental data were analyzed using OriginPro 2018 software.Simulation of kinetic curves according to the suggested mechanism was performed in the ChemMech program package [53], which performs minimization of deviation between experimental and calculated data.

DFT calculations
DFT calculations were performed using ORCA 5.0.4 software package [54].Geometries of truncated Cbl species were determined using the B3LYP functional and the def2-TZVP basis set.

Results and discussion
The addition of Angeli's salt to GSCbl at pH 7.4 results in changes in the UV-vis spectrum shown by Figure 2. The UV-vis spectrum of the compound resembles that of NOCbl generated during the reaction between H 2 OCbl and Angeli's salt [41].However, the position of the absorption maximum of the product generated in the course of the reaction between GSCbl and Angeli's salt (485 nm) is ca. 4 nm red-shifted in comparison with authentic NOCbl (481 nm [30]).To explain this difference, we studied the influence of pH on the UV-vis spectrum of species produced from GSCbl upon its reaction with Angeli's salt.We found that in an alkaline medium the UV-vis spectrum of the product exhibits maxima at ca. 320 and 480 nm.In contrast, new maxima emerge at 354, 413, and 531 nm upon acidification of the reaction medium (Figure 3(A)).The dependence of absorbance at 480 nm on pH exhibits an inflection point at pH 7.2 (Figure 3(A)).The use of signals inside the aromatic region of 1 H NMR spectra also aids in the identification of Cbl species.Species generated in alkaline medium exhibit signals at 6.27, 6.34, 6.80, 7.21, and 7.38 ppm, and product formed in weakly acidic solutions has signals at 6.22, 6.29, 6.43, 6.75, and 7.20 ppm (Figure 3(B)).Full 1 H NMR spectra are shown in Figure S1.Maxima in the UV-vis spectrum and signals in the 1 H NMR spectrum of Cbl species formed in the alkaline medium are very close to those of NOCbl (k max : 314 and 481 nm [30]; d: 6.26 [55], 6.34 [55], 6.76 [25], 7.20 [25] and 7.40 [25] ppm), and species produced in the weakly acidic medium can be assigned to the complex of Cbl(III) with nitrite (nitrocobalamin, NO 2 Cbl; k max : 354, 413 and 532 nm; d: 6.20, 6.28, 6.42, 6.74 and 7.20 ppm [48]).Thus, the main products of the reaction between GSCbl and Angeli's salt in acidic and alkaline solutions are NO 2 Cbl and NOCbl, respectively, whereas both species are formed in neutral solutions.
We determined the stoichiometry of the reaction between GSCbl and Angeli's salt at pH 7.4.Conversion of GSCbl to the mixture of NOCbl and NO 2 Cbl requires an almost 10-fold excess of Angeli's salt over GSCbl (Figure S2).In the case of H 2 OCbl, the formation of NOCbl proceeds in an equimolar mixture of H 2 OCbl and Angeli's salt [41].This can be explained by the lower reaction rate involving GSCbl, which implies a significant extent of decomposition of active reactant species (viz.HNO and/or Angeli's salt) prior to their interaction with GSCbl.
To identify species involved in the reaction with GSCbl, we examined the influence of nitrite, another product of Angeli's salt decomposition, and nitroxyl scavengers (viz.glutathione, TCEP, and ascorbic acid) on the reaction between GSCbl and Angeli's salt at pH 7.4.The addition of nitrite to GSCbl does not result in changes in the UV-vis spectrum for at least 1 h (Figure S3), indicating involvement of Angeli's salt or/and HNO in the reaction with GSCbl.The addition of nitrite slightly decreases the reaction rate of the reaction between GSCbl and Angeli's salt (Figure S4).It has been reported that NO 2 − is capable of binding with HNO leading to Angeli's salt regeneration and increasing its stability in a neutral medium [56].Thus, HNO can be one of the reactive species in this system; however, its contribution to the reaction is relatively low.This fact is supported by the influence of HNO scavengers on the studied reaction.Ascorbic acid is ca.10-fold less reactive toward HNO than TCEP and GSH [11].Thus, we used higher ascorbic acid concentrations than TCEP and GSH to reach a similar HNO scavenging effect.Note that GSCbl does not react with ascorbic acid, GSH and TCEP in neutral solutions.The impact of ascorbic acid on the interaction between GSCbl and Angeli's salt exhibited dissimilarities compared to that of TCEP and GSH.Specifically, the presence of ascorbic acid resulted in a modest reduction in the reaction rate, as seen in Figure S5.In contrast, induction periods are observed in the presence of GSH (Figure S6) and TCEP (Figure S7) in the system.GSH and TCEP can probably react not only with HNO generated upon decomposition of Angeli's salt but also with some intermediate species (e.g. a complex of Angeli's salt with GSCbl) that leads to significant inhibition of the reaction.In contrast, ascorbic acid acts solely as a trap for HNO.
Next, we studied the kinetics of the reaction between GSCbl and Angeli's salt.The typical kinetic curve of the reaction resembles the first-order function (Figure 2).However, simplified fitting of kinetic data using the exponential equation or application of the initial rate method in this case must be avoided due to numerous potential reactions in which initial reactants (viz.GSCbl and Angeli's salt) and decomposition products of Angeli's salt (viz.nitroxyl and nitrite) can be involved.In particular, GSCbl may react directly with Angeli's salt (reaction 1) and/or with nitroxyl (reaction 2) generated upon Angeli's salt decomposition (reaction 3).Monoprotonated and fully deprotonated Angeli's anions may possess different reactivity toward GSCbl; thus, reaction 4 (pK a1 ¼ 9.4 at 25.0 � C [51]) was included in the mechanism.HNO and NO 2 − can regenerate the initial Angeli's salt (reaction 5; k 4 ¼ 1.0 � 10 3 M −1 s −1 [56]).HNO is a shortliving molecule that undergoes dimerization (reaction 6; k 5 ¼ 8.0 � 10 6 M −1 s −1 [57]) and subsequent dehydration to give N 2 O.We also added to the reaction mechanism between free HNO and free GSH (reaction 7), which can be generated upon glutathionyl-ligand substitution by HNO or Angeli's salt.Although the rate constant for the reaction between HNO and GSH species with protonated thiol group is known [9-11], the rate constant for the process involving deprotonated thiol species (pK a2 ¼ 8.9 [58]; reaction 8) has not been reported.Apparently, thiolate species are more reactive toward HNO than thiols.However, further simulation indicated an almost negligible contribution of reaction 7 (with k 6 � 2.0 � 10 6 M −1 s −1 [9-11]) to the shape of the produced kinetic curves and the values of the determined rate constants.Though free GSH can be released from GSCbl upon its protonation, the protonation of sulfur atom in GSCbl occurs in a relatively strongly acidic medium (pK a ¼ 1.3 [59]), which is beyond the conditions used in this study.Thus, this process does not contribute to the mechanisms studied in the present work.HNO is capable of reacting with hydroxylamine [11,60], which can be formed upon HNO reduction by thiols [12].However, we found no influence of NH 2 OH on the reaction between GSCbl and Angeli's salt (Figure S8).Moreover, the quantity of NH 2 OH formed in this reaction, determined using the indooxine method (Figure S9), was very low (Table S1).Thus, the model did not include the reaction between HNO and NH 2 OH.In basic medium, HNO is deprotonated to NO − (reaction 9; pK a3 ¼ 11.5 [42]), whose behavior in reactions 5 and 6 is poorly understood.Thus, kinetic measurements were performed under conditions where HNO is the predominant form of nitroxyl in solution.
We utilized the ChemMech program to simulate kinetic curves to identify the nature of reactive species involved in GSCbl conversion to NOCbl and NO 2 Cbl and to determine the rate constants of these processes.During simulations, rate constants k 3 , k 4 , k 5 and k 6 were fixed, and k 1 and k 2 were varied.At pH 7.4, the lowest deviation between experimental and simulated data was reached by the inclusion of reactions of GSCbl with HNO and HN 2 O 3 − in the model (Figure 4).Using only the reaction between GSCbl and HNO provides poor fitting of experimental data, whereas considering only the reaction between GSCbl and HN 2 O 3 − gives relatively high-quality simulated data (Figure S10).The same situation, i.e. the predominant contribution of reaction 1 and low influence of reaction 2, was observed in acidic medium (Figure S11).On the contrary, in an alkaline medium, reaction 2 exhibits a high contribution to the mechanism (Figures S12-S16), and the rate constant of reaction 1 is indistinguishable from zero (Figures S14-S16).and GSNHO − (reaction 12), and the deprotonated form decomposes to initial reactants (reaction 13).Considering reactions 10-13, Equation ( 14) can be derived.Using Equation (11), values k 7 K 1 ¼ (3.5 ± 0.1) M −1 s −1 and pK a4 ¼ (8.1 ± 0.1) can be determined.
For the reaction between GSCbl and HNO, the dependence of the rate constant (k 2 ) on pH indicates an increase in k 2 upon alkalinization of the medium up to pH 10.3 (Figure 5(B)).HNO undergoes deprotonation under basic conditions (reaction 9; pK a3 ¼ 11.5 [42]) to give NO − , which can be more reactive toward GSCbl than HNO.Considering reactions 15 and 16, Equation ( 17) was derived.However, fitting the data provided by Figure 5(B) by Equation ( 17) with pK a ¼ 11.5 gives unsatisfactory results, and the best fit can be achieved with pK a ¼ (8.9 ± 0.2) (Figure 5(B)).Thus, the reaction between GSCbl and HNO includes another acid-base equilibrium.We assume the complexation between GSCbl and HNO (reaction 18) to give the GS(HNO)Cbl complex that can be further deprotonated to GS(NO − )Cbl (reaction 19).Protonated and deprotonated species undergo decomposition to NOCbl and GS − (H þ ) (reactions 20 and 21), and in the case of the deprotonated ones, the reaction proceeds more rapidly.Fitting the data provided by Figure 5(B) by Equation ( 17), the values k 8 (k 10 K 2 ) ¼ (3.6 ± 1.1)�10 3 M −1 s −1 and k 9 (k 11 K 2 ) ¼ (2.0 ± 0.2)�10 4 M −1 s −1 were determined.Further, we examined the formation of transient complexes between GSCbl and HNO or HN 2 O 3 − using DFT calculations.In these computations, the truncated Cbl models were employed, in which side amide chains were replaced by hydrogens and glutathionyl ligand was replaced by methylthiolate.In optimized models, axial bond lengths were comparable with those determined by X-ray diffraction analysis, i.e. calculated Co-X (the upper axial ligand) bond lengths are 2.290 (GSCbl model) and 1.859 (NOCbl model) Å, whereas the experimental values are 2.295 (GSCbl) [61] and 1.907 (NOCbl) [27] Å (Figure S18).The optimized geometries of GS(HNO)Cbl and GS(HN 2 O 3 − )Cbl complexes are provided in Figure 6.In the case of GS(HNO)Cbl, a distance of 2.908 Å between the H-atom of HNO and the sulfur atom of GSCbl is observed, which can be attributed to a weak complexation between reactants.The behavior of deprotonated nitroxyl versions in interaction with GSCbl depends on their spin state.In particular, 3 NO − acts similarly to HNO, i.e. calculations indicate a weak bonding between nitroxyl nitrogen and thiol sulfur atoms, whereas 1 NO − tends to strongly interact with the macrocycle (Figure S19).In comparison with the GS(HNO)Cbl model, the complexation between HN 2 O 3 − and GSCbl is weaker (Figure 6), i.e. the distance between oxygen (NO 2 motif of HN 2 O 3 − ) and sulfur atoms of GSCbl is above 4 Å, though the NO 2 motif of HN 2 O 3 − is located closer to the macrocycle (3.103 Å).
Interestingly, in the absence of glutathionyl ligand, the orientation of HN 2 O 3 − toward macrocycle is altered, i.e. pronounced binding between the nitrogen atom of the HON motif of Angeli's anion and cobalt ion (ca.2.0 Å bond) is predicted (Figure S20), which might precede earlier observed HNO transfer [41].In the case of Angeli's dianion (N 2 O 3 2-), its interaction with the upper side of the corrin plane is not predicted (Figure S21), and N 2 O 3 2-tends to form complexes with 5,6-dimethylbenzimidazole nucleotide (DMBI) (viz.with hydrogen atom which does not exist in a real GSCbl structure).Moreover, N 2 O 3 2-is a stable anion and does not act as a nitroxyl source, so its contribution to the reaction with GSCbl is unlikely.Finally, we tested the interaction of HNO and HN 2 O 3 − with the lower part of the corrin plane in GSCbl.In both cases, reactants are located remotely from Co-ion (Figure S22), and the direct displacement of DMBI by HNO or HN 2 O 3 − is unlikely.Nevertheless, the possibility of the reaction pathway involving preliminary DMBI dissociation and successive HNO or HN 2 O 3 − binding to the Co-ion exists.However, this pathway cannot explain the generation of different products (viz.nitrosyl and nitro-Cbls) depending on pH.Thus, DFT calculations suggest weak HNO/ 3 NO − bonding with the S-atom of GSCbl.Apparently, further Co-S bond elongation may increase the strength of the HNO/ 3 NO − -GSCbl interaction.In the case of HN 2 O 3 − , calculations predict a weak interaction between NO 2 -motif and macrocycle.
This facile orientation may lead to further NO 2 -motif binding with Co-ion upon elongation of the Co-S bond, which explains nitro-Cbl formation in weakly acidic and neutral media.

Conclusion
Reactions of nitroxyl with biological molecules have received attention in the literature; e.g. the reaction of HNO with thiols may constitute a significant pathway of its signaling.Metal-bound thiolate ligands are ubiquitous in nature, although their reactions with HNO are poorly understood.This work showed that reactions of HNO with bound thiolato ligands are feasible.In particular, glutathionylcobalamin, a tight complex of cobalamin with glutathione, can react directly with HNO and its donor, Angeli's salt.The process involving HNO proceeds in neutral and alkaline media and produces nitrosylcobalamin.It proceeds via transient complexation between reactants, and the generated complex is involved in an acid-base equilibrium.Protonated and deprotonated forms of this complex are further transformed to NOCbl and GSH, and the transformation of the deprotonated species occurs more rapidly.The process involving GSCbl and Angeli's salt is negligible in an alkaline medium and accelerated upon lowering pH.The product of this reaction is nitrocobalamin.The reaction involves complexation between GSCbl and HN 2 O 3 − and further decomposition of this complex to NO 2 Cbl and GSNHO − .At pH 7.4, the contribution of the pathway involving HNO has a slightly more pronounced contribution to the GSCbl conversion than the reaction with HN 2 O 3 − .The rate constant of the reaction between GSCbl and HNO at pH 7.4 is ca. 10 3 -times lower than that for free GSH; thus, this reaction is too slow to be biologically relevant.However, GSH binding may protect cobalamins from inactivation by HNO.
9; pK a3 ) Dependencies of rate constants for reactions of GSCbl with HN 2 O 3 − (k 1 ) and HNO (k 2 ) on pH are shown in Figure 5.In the case of the reaction between GSCbl with HN 2 O 3 − , the pH-dependence presents descending sigmoid profile with inflection point at pH 8.1 (Figure 5(A)).It is known that HN 2 O 3 − undergoes deprotonation in basic medium.However, the pK a1 of this equilibrium (i.e.pK a1 ¼ 9.4 at 25.0 � C [51]) is higher than 8.1.This fact can be explained by the complexation step between GSCbl and HN 2 O 3 − (reaction 10) that facilitates HN 2 O 3 − deprotonation (reaction 11).The protonated complex between GSCbl and HN 2 O 3 − further decomposes to nitrocobalamin