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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