Nitro-fatty acids modulate inflammatory and metabolic stress responses, thus displaying potential as new drug candidates. Herein, we evaluate the redox behavior of nitro-oleic acid (NO2-OA) and its ability to bind to the fatty acid transporter human serum albumin (HSA). The nitro group of NO2-OA underwent electrochemical reduction at -0.75 V at pH 7.4 in an aqueous milieu. Based on observations of the R-NO2 reduction process, the stability and reactivity of NO2-OA was measured in comparison to oleic acid (OA) as the negative control. These electrochemically-based results were reinforced by computational quantum mechanical modeling. DFT calculations indicated that both the C9-NO2 and C10-NO2 positional isomers of NO2-OA occurred in two conformers with different internal angles (69° and 110°) between the methyl- and carboxylate termini. Both NO2-OA positional isomers have LUMO energies of around -0.7 eV, affirming the electrophilic properties of fatty acid nitroalkenes. In addition, the binding of NO2-OA and OA with HSA revealed a molar ratio of ~7:1 [NO2-OA]:[HSA]. These binding experiments were performed using both an electrocatalytic approach and electron paramagnetic resonance (EPR) spectroscopy using 16-doxyl stearic acid. Using a Fe(DTCS)2 spin-trap, EPR studies also showed that the release of the nitro moiety of NO2-OA resulted in the formation of nitric oxide radical. Finally, the interaction of NO2-OA with HSA was monitored via Tyr and Trp residue electro-oxidation. The results indicate that not only non-covalent binding but also NO2-OA-HSA adduction mechanisms should be taken into consideration. This study of the redox properties of NO2-OA is applicable to the characterization of other electrophilic mediators of biological and pharmacological relevance.

Department of Chemistry Faculty of Science University of South Bohemia Branisovska 31 Ceske Budejovice 370 05 Czech Republic

Department of Medical Chemistry and Biochemistry Faculty of Medicine and Dentistry Palacky University Hnevotinska 3 Olomouc 775 15 Czech Republic

Department of Medical Chemistry and Biochemistry Faculty of Medicine and Dentistry Palacky University Hnevotinska 3 Olomouc 775 15 Czech Republic; The Czech Academy of Sciences Institute of Biophysics Kralovopolska 135 Brno 612 65 Czech Republic

Department of Pharmacology and Chemical Biology University of Pittsburgh School of Medicine Pittsburgh PA 15261 USA

Faculty of Physical Chemistry University of Belgrade Studentski trg 12 16 Belgrade Serbia

The Czech Academy of Sciences Institute of Biophysics Kralovopolska 135 Brno 612 65 Czech Republic

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Egea J., Fabregat I., Frapart Y.M., et al. European contribution to the study of ROS: a summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS) Redox Biol. 2017;13:94–162. PubMed PMC

Groeger A.L., Freeman B.A. Signaling actions of electrophiles: anti-inflammatory therapeutic candidates. Mol. Interv. 2010;10:39–50. PubMed PMC

Liebler D.C. Protein damage by reactive electrophiles: targets and consequences. Chem. Res. Toxicol. 2008;21:117–128. PubMed PMC

Tornqvist M., Fred C., Haglund J., Helleberg H., Paulsson B., Rydberg P. Protein adducts: quantitative and qualitative aspects of their formation, analysis and applications. J. Chromatogr. B. 2002;778:279–308. PubMed

Turell L., Vitturi D.A., Coitino E.L., Lebrato L., Moller M.N., Sagasti C., Salvatore S.R., Woodcock S.R., Alvarez B., Schopfer F.J. The chemical basis of thiol addition to nitro-conjugated linoleic acid, a protective cell-signaling lipid. J. Biol. Chem. 2017;292:1145–1159. PubMed PMC

Hansen R.E., Roth D., Winther J.R. Quantifying the global cellular thiol-disulfide status. Proc. Natl. Acad. Sci. U.S.A. 2009;106:422–427. PubMed PMC

Rubino F.M., Pitton M., Di Fabio D., Colombi A. Toward an “omic” physiopathology of reactive chemicals: thirty years of mass spectrometric study of the protein adducts with endogenous and xenobiotic compounds. Mass Spectrom. Rev. 2009;28:725–784. PubMed

Rappaport S.M., Li H., Grigoryan H., Funk W.E., Williams E.R. Adductomics: characterizing exposures to reactive electrophiles. Toxicol. Lett. 2012;213:83–90. PubMed PMC

Freeman B.A., Baker P.R., Schopfer F.J., Woodcock S.R., Napolitano A., d'Ischia M. Nitro-fatty acid formation and signaling. J. Biol. Chem. 2008;283:15515–15519. PubMed PMC

Rudolph V., Rudolph T.K., Schopfer F.J., Bonacci G., Woodcock S.R., Cole M.P., Baker P.R., Ramani R., Freeman B.A. Endogenous generation and protective effects of nitro-fatty acids in a murine model of focal cardiac ischaemia and reperfusion. Cardiovasc. Res. 2010;85:155–166. PubMed PMC

Vitturi D.A., Minarrieta L., Salvatore S.R., Postlethwait E.M., Fazzari M., Ferrer-Sueta G., Lancaster J.R., Jr., Freeman B.A., Schopfer F.J. Convergence of biological nitration and nitrosation via symmetrical nitrous anhydride. Nat. Chem. Biol. 2015;11:504–510. PubMed PMC

Vitturi D.A., Chen C.S., Woodcock S.R., Salvatore S.R., Bonacci G., Koenitzer J.R., Stewart N.A., Wakabayashi N., Kensler T.W., Freeman B.A., Schopfer F.J. Modulation of nitro-fatty acid signaling: prostaglandin reductase-1 is a nitroalkene reductase. J. Biol. Chem. 2013;288:25626–25637. PubMed PMC

Salvatore S.R., Vitturi D.A., Baker P.R., Bonacci G., Koenitzer J.R., Woodcock S.R., Freeman B.A., Schopfer F.J. Characterization and quantification of endogenous fatty acid nitroalkene metabolites in human urine. J. Lipid Res. 2013;54:1998–2009. PubMed PMC

Buchan G.J., Bonacci G., Fazzari M., Salvatore S.R., Gelhaus Wendell S. Nitro-fatty acid formation and metabolism. Nitric Oxide. 2018;79:38–44. PubMed PMC

Schopfer F.J., Vitturi D.A., Jorkasky D.K., Freeman B.A. Nitro-fatty acids: new drug candidates for chronic inflammatory and fibrotic diseases. Nitric Oxide. 2018;79:31–37. PubMed PMC

Schopfer F.J., Cipollina C., Freeman B.A. Formation and signaling actions of electrophilic lipids. Chem. Rev. 2011;111:5997–6021. PubMed PMC

Rudolph T.K., Freeman B.A. Transduction of redox signaling by electrophile-protein reactions. Sci. Signal. 2009;2:re7. PubMed PMC

Batthyany C., Schopfer F.J., Baker P.R., Duran R., Baker L.M., Huang Y., Cervenansky C., Branchaud B.P., Freeman B.A. Reversible post-translational modification of proteins by nitrated fatty acids in vivo. J. Biol. Chem. 2006;281:20450–20463. PubMed PMC

Cui T., Schopfer F.J., Zhang J., Chen K., Ichikawa T., Baker P.R., Batthyany C., Chacko B.K., Feng X., Patel R.P., Agarwal A., Freeman B.A., Chen Y.E. Nitrated fatty acids: endogenous anti-inflammatory signaling mediators. J. Biol. Chem. 2006;281:35686–35698. PubMed PMC

Kelley E.E., Batthyany C.I., Hundley N.J., Woodcock S.R., Bonacci G., Del Rio J.M., Schopfer F.J., Lancaster J.R., Jr., Freeman B.A., Tarpey M.M. Nitro-oleic acid, a novel and irreversible inhibitor of xanthine oxidoreductase. J. Biol. Chem. 2008;283:36176–36184. PubMed PMC

Awwad K., Steinbrink S.D., Fromel T., Lill N., Isaak J., Hafner A.K., Roos J., Hofmann B., Heide H., Geisslinger G., Steinhilber D., Freeman B.A., Maier T.J., Fleming I. Electrophilic fatty acid species inhibit 5-lipoxygenase and attenuate sepsis-induced pulmonary inflammation. Antioxidants Redox Signal. 2014;20:2667–2680. PubMed PMC

Schopfer F.J., Cole M.P., Groeger A.L., Chen C.S., Khoo N.K., Woodcock S.R., Golin-Bisello F., Motanya U.N., Li Y., Zhang J., Garcia-Barrio M.T., Rudolph T.K., Rudolph V., Bonacci G., Baker P.R., Xu H.E., Batthyany C.I., Chen Y.E., Hallis T.M., Freeman B.A. Covalent peroxisome proliferator-activated receptor gamma adduction by nitro-fatty acids: selective ligand activity and anti-diabetic signaling actions. J. Biol. Chem. 2010;285:12321–12333. PubMed PMC

Zhang J., Villacorta L., Chang L., Fan Z., Hamblin M., Zhu T., Chen C.S., Cole M.P., Schopfer F.J., Deng C.X., Garcia-Barrio M.T., Feng Y.H., Freeman B.A., Chen Y.E. Nitro-oleic acid inhibits angiotensin II-induced hypertension. Circ. Res. 2010;107:540–548. PubMed PMC

Kansanen E., Bonacci G., Schopfer F.J., Kuosmanen S.M., Tong K.I., Leinonen H., Woodcock S.R., Yamamoto M., Carlberg C., Yla-Herttuala S., Freeman B.A., Levonen A.L. Electrophilic nitro-fatty acids activate NRF2 by a KEAP1 cysteine 151-independent mechanism. J. Biol. Chem. 2011;286:14019–14027. PubMed PMC

Gorczynski M.J., Huang J., Lee H., King S.B. Evaluation of nitroalkenes as nitric oxide donors. Bioorg. Med. Chem. Lett. 2007;17:2013–2017. PubMed

Mata-Perez C., Sanchez-Calvo B., Begara-Morales J.C., Carreras A., Padilla M.N., Melguizo M., Valderrama R., Corpas F.J., Barroso J.B. Nitro-linolenic acid is a nitric oxide donor. Nitric Oxide. 2016;57:57–63. PubMed

Schopfer F.J., Lin Y., Baker P.R., Cui T., Garcia-Barrio M., Zhang J., Chen K., Chen Y.E., Freeman B.A. Nitrolinoleic acid: an endogenous peroxisome proliferator-activated receptor gamma ligand. Proc. Natl. Acad. Sci. U.S.A. 2005;102:2340–2345. PubMed PMC

Lima E.S., Bonini M.G., Augusto O., Barbeiro H.V., Souza H.P., Abdalla D.S. Nitrated lipids decompose to nitric oxide and lipid radicals and cause vasorelaxation. Free Radic. Biol. Med. 2005;39:532–539. PubMed

Schopfer F.J., Baker P.R., Giles G., Chumley P., Batthyany C., Crawford J., Patel R.P., Hogg N., Branchaud B.P., Lancaster J.R., Jr., Freeman B.A. Fatty acid transduction of nitric oxide signaling. Nitrolinoleic acid is a hydrophobically stabilized nitric oxide donor. J. Biol. Chem. 2005;280:19289–19297. PubMed

Schopfer F.J., Batthyany C., Baker P.R., Bonacci G., Cole M.P., Rudolph V., Groeger A.L., Rudolph T.K., Nadtochiy S., Brookes P.S., Freeman B.A. Detection and quantification of protein adduction by electrophilic fatty acids: mitochondrial generation of fatty acid nitroalkene derivatives. Free Radic. Biol. Med. 2009;46:1250–1259. PubMed PMC

Baker L.M., Baker P.R., Golin-Bisello F., Schopfer F.J., Fink M., Woodcock S.R., Branchaud B.P., Radi R., Freeman B.A. Nitro-fatty acid reaction with glutathione and cysteine. Kinetic analysis of thiol alkylation by a Michael addition reaction. J. Biol. Chem. 2007;282:31085–31093. PubMed PMC

Faine L.A., Cavalcanti D.M., Rudnicki M., Ferderbar S., Macedo S.M., Souza H.P., Farsky S.H., Bosca L., Abdalla D.S. Bioactivity of nitrolinoleate: effects on adhesion molecules and CD40-CD40L system. J. Nutr. Biochem. 2010;21:125–132. PubMed

Mata-Perez C., Sanchez-Calvo B., Padilla M.N., Begara-Morales J.C., Valderrama R., Corpas F.J., Barroso J.B. Nitro-fatty acids in plant signaling: new key mediators of nitric oxide metabolism. Redox Biol. 2017;11:554–561. PubMed PMC

Palecek E., Postbieglova I. Adsorptive stripping voltammetry of biomacromolecules with transfer of the adsorbed layer. J. Electroanal. Chem. 1986;214:359–371.

Laemmli U.K. Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature. 1970;227:680–685. PubMed

Ornstein L. Disc electrophoresis .I. Background and theory. Ann. NY Acad. Sci. 1964;121:321. PubMed

Davis B.J. Disc electrophoresis .2. Method and application to human serum albumins. Ann. NY Acad.Sci. 1964;121:404. PubMed

Scalmani G., Frisch M.J. Continuous surface charge polarizable continuum models of solvation. I. General formalism. J. Chem. Phys. 2010;132:114110. PubMed

Frisch M.J., Trucks G.W., Schlegel H.B., et al. Gaussian, Inc.; Wallingford CT: 2016. Gaussian 16, Revision B.01.

Petitpas I., Grune T., Bhattacharya A.A., Curry S. Crystal structures of human serum albumin complexed with monounsaturated and polyunsaturated fatty acids. J. Mol. Biol. 2001;314:955–960. PubMed

Mascarenhas R.J., Namboothiri I.N., Sherigara B.S., Mahadevan K.M. The electrochemical behaviour of novel multifunctional alpha-hydroxymethylated nitroalkenes at glassy carbon and wax impregnated carbon paste electrodes. Croat. Chem. Acta. 2007;80:53–59.

Squella J.A., Bollo S., Nunez-Vergara L.J. Recent developments in the electrochemistry of some nitro compounds of biological significance. Curr. Org. Chem. 2005;9:565–581.

Kraiya C., Singh P., Todres Z.V., Evans D.H. Voltammetric studies of the reduction of cis-and trans-α-nitrostilbene. J. Electroanal. Chem. 2004;563:171–180.

Bard A.J., Faulkner L.R. John Willey & Sons; New York, USA: 2001. Electrochemical Methods: Fundamentals and Applications.

Havran L., Billova S., Palecek E. Electroactivity of avidin and streptavidin. Avidin signals at mercury and carbon electrodes respond to biotin binding. Electroanalysis. 2004;16:1139–1148.

Kaneko F., Yano J., Sato K. Diversity in the fatty-acid conformation and chain packing of cis-unsaturated lipids. Curr. Opin. Struct. Biol. 1998;8:417–425. PubMed

Kobayashi M., Kaneko F., Sato K., Suzuki M. Vibrational spectroscopic study on polymorphism and order-disorder phase transition in oleic acid. J. Phys. Chem. 1986;90:6371–6378.

Tandon P., Forster G., Neubert R., Wartewig S. Phase transitions in oleic acid as studied by X-ray diffraction and FT-Raman spectroscopy. J. Mol. Struct. 2000;524:201–215.

Mishra S., Chaturvedi D., Kumar N., Tandon P., Siesler H.W. An ab initio and DFT study of structure and vibrational spectra of gamma form of oleic acid: comparison to experimental data. Chem. Phys. Lipids. 2010;163:207–217. PubMed

Ye C., Li M., Luo J., Chen L., Tang Z., Pei J., Jiang L., Song Y., Zhu D. Photo-induced amplification of readout contrast in nanoscale data storage. J. Mater. Chem. 2012;22:4299–4305.

Doneux T., Ostatna V., Palecek E. On the mechanism of hydrogen evolution catalysis by proteins: a case study with bovine serum albumin. Electrochim. Acta. 2011;56:9337–9343.

Havlikova M., Zatloukalova M., Ulrichova J., Dobes P., Vacek J. Electrocatalytic assay for monitoring methylglyoxal-mediated protein glycation. Anal. Chem. 2015;87:1757–1763. PubMed

Vacek J., Svrckova M., Zatloukalova M., Novak D., Proskova J., Langova K., Galuszkova D., Ulrichova J. Electrocatalytic artificial carbonylation assay for observation of human serum albumin inter-individual properties. Anal. Biochem. 2018;550:137–143. PubMed

Brabec V., Mornstein V. Electrochemical-behavior of proteins at graphite-electrodes.2. electrooxidation of amino-acids. Biochim. Biophys. Acta. 1980;12:159–165. PubMed

Brabec V., Mornstein V. Electrochemical-behavior of proteins at graphite-electrodes.1. electrooxidation of proteins as a new probe of protein-structure and reactions. Biophys. Chem. 1980;625:43–50. PubMed

Domotor O., Rathgeb A., Kuhn P.S., Popovic-Bijelic A., Bacic G., Enyedy E.A., Arion V.B. Investigation of the binding of cis/trans-[MCl4(1H-indazole)(NO)](-) (M = Ru, Os) complexes to human serum albumin. J. Inorg. Biochem. 2016;159:37–44. PubMed

Gurachevsky A., Shimanovitch E., Gurachevskaya T., Muravsky V. Intra-albumin migration of bound fatty acid probed by spin label ESR. Biochem. Biophys. Res. Commun. 2007;360:852–856. PubMed

Lagercrantz C., Larsson T., Karlsson H., Setaka M. Quantitative studies on competitive ligand-binding to bovine serum-albumin by use of spin label 5-doxyl dodecanoic acid. Eur. J. Biochem. 1978;83:197–203. PubMed

Lagercrantz C., Setaka M. Some binding properties of human-serum albumin as studied by spin labels 12-doxyl stearic acid and its methyl-ester. Acta Chem. Scand. B. 1975;29:397–398. PubMed

Morrisett J.D., Pownall H.J., Gotto A.M. Bovine serum albumin. Study of the fatty acid and steroid binding sites using spin-labeled lipids. J. Biol. Chem. 1975;250:2487–2494. PubMed

Pavicevic A., Luo J.H., Popovic-Bijelic A., Mojovic M. Maleimido-proxyl as an EPR spin label for the evaluation of conformational changes of albumin. Eur. Biophys. J. 2017;46:773–787. PubMed

Pavicevic A.A., Popovic-Bijelic A.D., Mojovic M.D., Susnjar S.V., Bacic G.G. Binding of doxyl stearic spin labels to human serum albumin: an EPR study. J. Phys. Chem. B. 2014;118:10898–10905. PubMed

Perkins R.C., Abumrad N., Balasubramanian K., Dalton L.R., Beth A.H., Park J.H., Park C.R. Equilibrium binding of spin-labeled fatty acids to bovine serum albumin: suitability as surrogate ligands for natural fatty acids. Biochemistry. 1982;21:4059–4064. PubMed

Ruf H.H., Gratzl M. Binding of nitroxide stearate spin labels to bovine serum-albumin. Biochim. Biophys. Acta. 1976;446:134–142. PubMed

Bhattacharya A.A., Grune T., Curry S. Crystallographic analysis reveals common modes of binding of medium and long-chain fatty acids to human serum albumin. J. Mol. Biol. 2000;303:721–732. PubMed

Peters T. Academic Press; San Diego: 1995. 3 - Ligand Binding by Albumin. All about Albumin; pp. 76–132.

Snyder N.W., Golin-Bisello F., Gao Y., Blair I.A., Freeman B.A., Wendell S.G. 15-Oxoeicosatetraenoic acid is a 15-hydroxyprostaglandin dehydrogenase-derived electrophilic mediator of inflammatory signaling pathways. Chem. Biol. Interact. 2015;234:144–153. PubMed PMC

Villacorta L., Chang L., Salvatore S.R., Ichikawa T., Zhang J.F., Petrovic-Djergovic D., Jia L.Y., Carlsen H., Schopfer F.J., Freeman B.A., Chen Y.E. Electrophilic nitro-fatty acids inhibit vascular inflammation by disrupting LPS-dependent TLR4 signalling in lipid rafts. Cardiovasc. Res. 2013;98:116–124. PubMed PMC

Woodcock C.S.C., Huang Y., Woodcock S.R., Salvatore S.R., Singh B., Golin-Bisello F., Davidson N.E., Neumann C.A., Freeman B.A., Wendell S.G. Nitro-fatty acid inhibition of triple-negative breast cancer cell viability, migration, invasion, and tumor growth. J. Biol. Chem. 2018;293:1120–1137. PubMed PMC

Kansanen E., Jyrkkanen H.K., Volger O.L., Leinonen H., Kivela A.M., Hakkinen S.K., Woodcock S.R., Schopfer F.J., Horrevoets A.J., Yla-Herttuala S., Freeman B.A., Levonen A.L. Nrf2-dependent and -independent responses to nitro-fatty acids in human endothelial cells: identification of heat shock response as the major pathway activated by nitro-oleic acid. J. Biol. Chem. 2009;284:33233–33241. PubMed PMC

Li S.D., Chang Z.Y., Zhu T.Q., Villacorta L., Li Y.X., Freeman B.A., Chen Y.E., Zhang J.F. Transcriptomic sequencing reveals diverse adaptive gene expression responses of human vascular smooth muscle cells to nitro-conjugated linoleic acid. Physiol. Genom. 2017;50:287–295. PubMed PMC

Fazzari M., Vitturi D.A., Woodcock S.R., Salvatore S.R., Freeman B.A., Schopfer F.J. Electrophilic fatty acid nitroalkenes are systemically transported and distributed upon esterification to complex lipids. J. Lipid Res. 2019;60:388–399. PubMed PMC

Fazzari M., Khoo N.K.H., Woodcock S.R., Jorkasky D.K., Li L.H., Schopfer F.J., Freeman B.A. Nitro-fatty acid pharmacokinetics in the adipose tissue compartment. J. Lipid Res. 2017;58:375–385. PubMed PMC

Khoo N.K.H., Li L.H., Salvatore S.R., Schopfer F.J., Freeman B.A. Electrophilic fatty acid nitroalkenes regulate Nrf2 and NF-kappa B signaling: a medicinal chemistry investigation of structure-function relationships. Sci. Rep. 2018;8(16) PubMed PMC

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