Characterization of a Water Soluble Iron-Nitroxyl Porphyrin Complex

AGOSTINA M. MAZZEO; JUAN PELLEGRINO; FABIO DOCTOROVICH

Chascomús

Workshop; 4th Latin American Meeting on Biological Inorganic Chemistry; 2014

Resumen: INTRODUCTION Interest in HNO (azanone according to IUPAC; usually nitroxyl) has increased in the last decade due to its remarkable chemical properties and increasing biological relevance.1 In addition, iron-nitroxyl complexes ({FeNO}8, according to the Enemark-Feltham notation)2 are important intermediates in NO and nitrite-reducing enzymes in bacteria and fungi, which catalyze important processes related to the biogeochemical cycle of nitrogen. The increasing number of reports on {FeNO}8 complexes have contributed to the spectroscopic characterization of this initially elusive species but still much more investigation is needed to elucidate mechanistic issues.3 We have recently reported a very stable {FeNO}8 complex, that could be isolated and characterized by UV-Vis, IR and 15N NMR, using the perhalogenated porphyrin TFPPBr8.4 Nevertheless, attempts to protonate the species to give the expected {FeHNO}8 complex were unsuccessful, disproportionating to the {FeNO}7 complex and H2. It has been considered that the protonated adduct is stabilized by amino acids, as in protein heme complexes,5 and bulky substituents, thought to provide steric protection that suppress the disproportionation reaction.6 However, the stability reported for the [Fe(CN)5(HNO)]3? complex7 leads us to consider that other factors, like hydrogen bonds with water, could stabilize the Fe-HNO moiety. In this work we obtained the {FeNO}8 adduct of the water-soluble porphyrin TPPS, which has been widely used in previous works on NO bioinorganic chemistry. So far, the product has been characterized by UV-Vis.   EXPERIMENTAL METHODS [FeIII(TPPS)]3? was synthesized  from the demetallated commercial porphyrin and FeSO4, according to methods described in literature.8 An excess of a sodium dithionite (Na2S2O4) solution (pH 12) was added to a solution of [FeIII(TPPS)]3? in a pH 6 NaH2PO4/Na2HPO4 buffer to obtain [FeII(TPPS)]4?. Afterwards, the {FeNO}7 complex was obtained by bubbling NO gas into the solution. NO gas was generated using a finely divided mixture of FeSO4.7H2O, NaBr and NaNO29 and was passed through a NaOH column to remove higher oxides. Once the {FeNO}7 precursor was obtained, an excess of the dithionite solution was added to obtain the {FeNO}8 complex. All solutions used were previously thoroughly degassed and the reactions were carried out in a screw-capped UV-Vis quartz cuvette with a septum in a UV-Vis spectrophotometer that allows a continuous argon flow.   RESULTS AND DISCUSSION Figure 1 shows the UV-vis spectral changes upon reduction of the [FeII(TPPS)NO]4? complex.     The reaction sequence to obtain the {FeNO}8 complex is shown in equations 1-3. The reducing agent was in both cases sodium dithionite, added in excess, since it quickly decomposes at the pH 6 of the porphyrin solution.     The same spectral changes were previously reported for the reduction of [FeII(TPPS)NO]4? by benzophenone ketyl radicals in a pH 6 phosphate buffer, through a much more complicated laser photolysis procedure.10 Under those conditions, the {FeNO}8 complex reoxidizes to the {FeNO}7 species with a half-life of about 2 s. Moreover, that method prevents the complete conversion from {FeNO}7 to {FeNO}8 due to partial photodissociation of NO in the precursor. In contrast, in our experiment, a full conversion was achieved and a much slower reoxidation was observed (with a half-life of several minutes). At this moment we cannot establish the identity of the oxidizing agent responsible for the reoxidation of the {FeNO}8 complex. One hypothesis proposes an oxygen leak in the system. Another possibility would be a reaction with the sulfite produced by dithionite reduction or with any other decomposition product, since dithionite is known to decompose in many different species in non-basic solutions. For this reason other reductants will be tried such as Cr(II) EDTA, that have been previously used to obtain a protein {FeNO}8 complex.5 A third hypothesis is that the protons are reduced to hydrogen, though that was not observed in the [Fe(CN)5(HNO)]3? complex. On the other hand, it has not been possible so far to determine whether the {FeNO}8 complex is protonated or not at the pH of the experiment.   CONCLUSION We have successfully obtained, using a straightforward procedure, a water-soluble {FeNO}8 porphyrin complex with greater stability than previously reported. At the time of this presentation, the experiments were carried out only in the UV-vis concentration range. We are presently attempting to optimize the reaction conditions in order to bring the experiment to a higher concentration scale. This will allow further characterization of the product by 1H NMR and IR, which have been proved to be the essential techniques for the unambiguous characterization of {FeHNO}8 complexes.11 Depending on the stability of the product, its reactivity with biologically relevant substrates, such as NO, could be studied. Another interesting issue that remains to be determined is the pKa associated to the process of protonation of the {FeNO}8 complex.   REFERENCES   Doctorovich, et al., Coord. Chem. Rev. (2011), 255:2764-2784. Enemark, J. H. et al., Coord. Chem. Rev. (1974), 13:339?406. Speelman, A. L. et al., Acc. Chem. Res. (2014), 47:1106?1116. Pellegrino, J, et al., J. Am. Chem Soc. (2010), 132:989-995. Lin, R. et al., J. Am. Chem. Soc. (2000) 122:2393?2394. Goodrich, L. E. et al., Inorg. Chem (2013). Montenegro, A. C. et al., Angew. Chemie Int. Ed. (2009), 48:4213−4216. Barley, M. H. et al., Inorg. Chem (1987), 26:1746-1750. Mason, J. et al., Chem. Rev. (2002), 102:913-934. Seki, H. et al., J. Chem. Soc., Faraday Trans. (1996), 92:2579?2583. Farmer, P. J. et al., J. Inorg. Biochem. (2005), 99:166-184.   ACKNOWLEDGMENTS The authors would like to thank CONICET, UBA and ANPCyT for providing financial support to this project.