Nitric oxide is reduced to HNO by proton-coupled nucleophilic addition (PCNA) of ascorbic acid, tyrosine, and other alcohols. A new route for HNO formation in biological media?

MARTINA MUÑOZ; SEBASTIÁN A. SUÁREZ; NICOLÁS I. NEUMAN; LUCÍA ÁLVAREZ; JAN MILJKOVIC; CARLOS BRONDINO; DAMIÁN E. BIKIEL; IVANA IVANOVIC-BURMAZOVIC; MILOS R. FILIPOVIC; MARCELO A. MARTÍ; FABIO DOCTOROVICH

Chascomús

Encuentro; IV LABIC Fourth Latin American Meeting on Biological Inorganic Chemistry V WOQUIBIO Fifth Workshop on Bioinorganic Chemistry; 2014

UBA, UNR, CONICET, AGENCIA, Workshop Argentino de Química Bioinorgánica

O14.-Nitric oxide is reduced to HNO by proton-coupled nucleophilic addition (PCNA) of ascorbic acid, tyrosine, and other alcohols. A new route for HNO formation in biological media? Martina Muñoz,? Sebastián A. Suarez,? Nicolás Neuman,?ǂ Lucia Alvarez,? Jan Miljkovic,# Carlos Brondino,ǂ Damián E. Bikiel,? Ivana Ivanovic-Burmazovic,# Milos R. Filipovic,# Marcelo A. Martí,?? Fabio Doctorovich?*. ? Departamento de Química Inorgánica, Analítica y Química Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina / INQUIMAE-CONICET. ? Departamento de Química Biológica, FCEN, UBA, Argentina ǂ Departamento de Física, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Santa Fe, Argentina. # Department of Chemistry and Pharmacy, Friedrich-Alexander University Erlangen-Nuremberg, Germany tini.m2@hotmail.com INTRODUCTION The key role of nitric oxide (NO) in several biological processes seems nowadays to be well established. However, it should be considered that azanone (HNO, nitroxyl), the one electron reduction product of NO, could also be involved in some of these processes. The key pitfall for a full understanding of NO/HNO chemistry has been the lack of direct and specific methods to detect HNO. However, in the last few years several detection and trapping methods have been developed,(1?8) which opened the opportunity for detailed studies of its biological relevant reactions. In this context, one of the most important yet unanswered questions is whether HNO is produced in-vivo or not. One of the possible routes concerns chemical or enzymatic reduction of NO. In the present work we have taken advantage of a highly selective and sensitive HNO trapping and quantification method, to show that NO is reduced to HNO by several physiological moderate reductants, like ascorbic acid (AA), phenol (Ph-OH), hydroquinone (HQ) and tyrosine (Tyr). EPR evidence for the formation of the corresponding radicals has also been found. EXPERIMENTAL METHODS NO was generated anaerobically by dropwise addition of degassed water to a mixture of 4 g NaNO2, 8.5 g FeSO4 and 8.5 g NaBr. The produced NO was passed through a NaOH solution to remove higher oxides and bubbled into degassed water in order to get a saturated solution of NO ([NO] = 2 mM). Amperometric measurements of HNO concentration were carried out with our previously described method based on a gold working electrode modified with a monolayer of cobalt porphyrin with 1-decanethiol covalently attached.(9, 10) The method has been demonstrated to be specific for HNO, showing no interference or spurious signal due to the presence of NO, O2, NO2 and other RNOS. EPR measurements X-band CW-EPR spectra of liquid mixtures of sodium ascorbate, hydroquinone or L-tyrosine and NO in buffer were acquired at room temperature using a Bruker EMX-Plus spectrometer with a rectangular cavity and a flat quartz cell. Macrophages culture: We used RAW 264.7 macrophages (Mouse leukaemic monocyte macrophage cell line). RESULTS AND DISCUSSION In Figure 1 we present the amperometric signal vs. time plot after the addition of NO (2 μM) to a solution of 2 μM AA. The increase in the current following addition of AA clearly evidences HNO formation. From the Log [C] vs. Log (rate) plot we confirmed that reaction is first order in both reactants. These plots also allow determination of an effective bimolecular reaction rate constant (keff) corresponding to: ROH + NO => HNO + RO* (Reaction 1) v = keff [ROH] [NO] Figure 1. A) Amperometric signal vs time plot after the addition of NO (2 μM) to a solution of 2 μM AA (left axis: [HNO] after calibration, right axis: measured current). B) Time dependence of the peak-to-peak amplitude of the low-field peak of the ascorbyl signal. Inset: EPR spectra of solutions of ascorbate (1 mM) alone and with NO (1 mM). As expected, in the absence of either reactant, no HNO is detected. Similar results were obtained with the other alcohols resulting keff are reported in Table 1. The data shows that both diols (HQ and AA) react >10 times faster than phenols, with AA being the fastest. The initial products of the reaction of NO with the alcohols are, as already mentioned, unstable and highly reactive. Thus, further reactions are expected to occur. HNO main sink is expected to be its dimerization and reaction with NO, yielding the stable products N NO2 ?. Table 1. Amounts of N2O and nitrite obtained for the reactions of H* donors with NO Compounda keff (M-1s-1)b NO2  (μmol)c N2O (μmol)c NO AA 83 (16) 3.2 1.3 Y 1 3.7 1.6 Ph 2 4.4 1.0 HQ 15 (4) 2.9 1.0 b Determined from the slope of the electrode signal. In brackets, determined from EPR signal c Based on the initial μmol) of NO. Ab-initio Mechanistic Analysis. As an example the results for AA are presented in Scheme 1. The calculations show that NO reaction with AA is endergonic yielding a possible ONOH intermediate which then decays to HNO and AA radical. This intermediate is consistent with one of the peaks observed in the mass spec = 207.02). HNO dimerization overcompensates the energy resulting in an overall negative free energy balance for the global reaction, which for AA is: AA + 2NO => DHA + N2O + H M f ted. he and the rization N2O and O NO2  : N2O N2O Yieldc 2.5 7% 2.3 8% 4.4 5% 2.9 5% amount (20 or spectrum (m/z . O H2O Scheme 1 ? Ab-initio Mechanistic Analysis. Energy values report in kcal/mol. CONCLUSION The present work presents clear evidence of a possible chemical biology HNO source, resulting from reaction of NO with rich aromatic alcohols tyrosine, and hydroquinone. REFERENCES 1. M. R. Cline, et al, Free Radic. Biol. Med. (2011) 50, 1274?9. 2. J. A. Reisz, et al, J. Am. Chem. Soc. (2011) 133, 11675?85. 3. S. Donzelli, et al, Free Radic. Biol. Med. (2008) 45, 578?84. 4. S. A. Suárez, et al, Polyhedron (2007) 26, 4673?9. 5. K. P. Dobmeier, et al, Anal. 1247?54. 6. M. A. Martí, et al, J. Am. Chem. Soc. (2005) 127, 4680?4. 7. Y. Zhou, et al, Org. Lett. (2011) 13, 2357 8. F. Doctorovich, et al, Coord. C (2011) 255, 2764?84. 9. S. A. Suárez, et al, Inorg. 6955?66. 10. S. A. Suárez, et al, Anal 10262-69. ACKNOWLEDGMENTS This work was financially supported by UBA, ANPCyT, CONICET and ByB Foundation.