FINE CONTROL OF THE STUDY OF INORGANIC SULFIDES REACTITY ON HEME COMPOUNDS (ALL THAT STINKS IS NOT SULFIDE)

BIEZA, SILVINA ANDREA; BOUBETA, FERNANDO MARTÍN; FEIS, ALESSANDRO; SMULEVICH, GIULIETTA; ESTRIN, DARÍO A.; BOECHI, LEONARDO; BARI, SARA ELIZABETH

Chascomús

Workshop; Fourth Latin American Meeting on Biological Inorganic Chemistry and FIfth Workshop on Bioinorganic Chemistry; 2014

Fine Control of the Study of Inorganic Sulfides Reactivity on Heme Compounds (All that Stinks is not Sulfide)   Silvina A. Bieza1, Fernando Boubeta1, Alessandro Feis3, Giulietta Smulevich3, Darío A. Estrín1, Leonardo Boechi2, and Sara E. Bari1 1Departamento de Química Inorgánica, Analítica y Química Física/ INQUIMAE-CONICET, 2Instituto del Cálculo, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina. 3Dipartimento di Chimica ?Ugo Schiff?, Universita' di Firenze, Italy. sbieza@qi.fcen.uba.ar     INTRODUCTION The endogenous production of hydrogen sulfide (H2S) impacts in several physiological processes1,2. It is known that among its biological targets are the protein thiols or the heme group of hemeproteins. Concerning the heme targets, certain ferric hemeproteins have been proved to bind sulfide species with a very high affinity3,4. There have also been reported some examples where the ferrous hemeprotein is the product of the reaction with sulfides5,6. The regulation and mechanisms of these reactions are unknown, and the analysis is somehow obscured by the purity of the source of sulfide.7 In an attempt to study the fundamentals of the chemical reactivity of inorganic sulfide towards hemeproteins, we selected a minimalist model, the undecapeptide microperoxidase MP11, which has a heme b (FeIII protoporphyrinate IX) moiety covalently attached to the peptide, and retains the interaction with the metal to a proximal histidine. The immediate and inexpensive sources for inorganic sulfides were Na2S.9H2O or NaHS.2H2O. We started working with NaHS.2H2O and had some encouraging preliminar results that soon turned into frustrating, erratic results. Herein, we present the results of the study of the reactivity of MP11 towards freshly prepared H2S or the anhydrous Na2S, that allow us to obtain reproducible experiments, analyzed under the light of UV-Vis and resonance Raman (RR) spectroscopies. The interpretation of the results required a detailed computational picture of the role of the aminoacids in the stabilization of the reaction product. The results presented herein are relevant to the exclusive role of the proximal histidine in the stabilization of ferric-sulfide complexes, FeIII-S.     EXPERIMENTAL METHODS FeIIINAcMP11 was prepared according to published procedures, from FeIIIMP11 (Sigma-Aldrich). Solutions of [FeIIINAcMP11] ranged from 10-6 to 10-5M, (buffer PO43-, 0.1M), and were deareated under argon. H2S was prepared from Na2S.9H2O and H3PO4 using Schlenk equipment, and transferred with gas-tight syringes. Anhydrous Na2S (Sigma Aldrich), was stored in an inert glove box. In a typical experiment (25ºC), 100 mL (1 atm) of H2S (g) were added to a solution of the FeIIINAcMP11 (2.5 mL). Stoichiometric amounts of the Na2S were dissolved in degassed buffer, and manipulated under inert atmosphere. UV-Vis spectra were recorded on a HP8453 spectrophotometer. The light source for RR spectroscopy was a Kr+ laser (Coherent, Innova 300C). The back-scattered light from a slowly rotating NMR tube was collected and focused into a triple spectrometer, equipped with a liquid N2 cooled CCD detector (Roper Sci., Princeton Instr.). MD simulations (7 x 200ns) of MP11FeIII(SH) where performed at neutral pH,  TIP3P  water as solvation model, using Amber Force Field, Berendsen Thermosthat, T=300K, and a time step of 1 fs. In order to avoid sampling issues, also accelerated MD simulations from different MD snapshots where performed. Na2S. 9H2O, NaHS.2H2O, 1-Methylimidazole, and cysteine were also from Sigma Aldrich.   RESULTS AND DISCUSSION The absorption spectra recorded after the addition of gaseous H2S, to a deoxygenated solution of FeIIINAcMP11 at pH 6.8 reveals three bands with maxima at 412 nm (lmax), 536 nm and 568 nm are observed. The calculated ratio H2S:MP11 ca. 100, could not be assessed in the solutions, but excess amounts of the sulfide could be observed at 230 nm. The final spectrum exhibits the features of low-spin state FeIII complexes, resembling that of the corresponding hydroxo complex, only obtained above pH 9,8 or the spectra obtained after the addition of alkylthiols or L-cysteine.9,10 The anhydrous Na2S allowed the stoichiometric analysis of the reaction and the evaluation of the reaction rate. The formation of negligible amounts of the FeIINAcMP11, was only observed for ratios above 1:25. The use of Na2S. 9H2O or NaHS.2H2O, as mentioned, yielded erratic results, with enhanced formation of the FeII form in most of the experiments. RR spectroscopy was performed on the 412 nm species, and compared to the FeIIINAcMP11. A comparison with the unbound form reveals the formation of a strong band at 366 cm-1, in analogy with other low spin FeIII-sulfide adducts, and is tentatively assigned to the Fe-S stretching mode.11,12 This frequency seems to characterize the Fe-S(inorganic) stretching mode, as the binding of cysteine does not show this frequency. During the time scale of our simulations, no residues were observed to interact with the coordinated [(HS-)FeIIIMP11], predicting even less likely interactions for the real sample, FeIIINacMP11. Accelerated MD simulations, revealed the same results.13 The simulations also revealed a dynamic H-bonding interaction between the Nd of the proximal His19 and the carboxyl group of Thr19. Although H-bonding induces rotation of this proximal histidine, our calculations show that the imidazole ring maintains a staggered conformation with respect to the heme. This computational picture highlights that the stability of complex formed after the addition of H2S or Na2S to NAcMP11 is attained even in the absence of the assistance of distal aminoacids. Selected reactivity tests, as the reaction with 1-methylimidazole, further support the ferric nature of the complex, and are currently assisting the determination of kinetic constants.   CONCLUSION FeIIINAcMP11 provides a model for the evaluation of proximal effects on the formation of hexacoordinated complexes, accepting a sulfide species as the sixth ligand devoid of distal mechanisms of stabilization. The formation of a moderately stable Fe-S complex, tentatively NAcMP11FeIII(SH-), supports the key role of the proximal histidine in the binding of inorganic sulfide species to hemoglobins. A special attention should be placed on the source of inorganic sulfides, as regular contaminants can lead to erratic and contradicting results.   REFERENCES 1. Abe, K.; Kimura, H. J. Neurosci. 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