REACTIVITY OF PENTACOORDINATE HEMEPROTEIN MODELS IN SODIUM DODECYL SULFATE MICELLES

BIEZA, SILVINA ANDREA; DIZ, VIRGINIA; ESTRIN, DARÍO A.; BARI, SARA ELIZABETH

Chascomús

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

Reactivity of Pentacoordinate Hemeprotein Models in Sodium Dodecyl Sulfate Micelles   Silvina A. Bieza, Virginia Diz, Darío A. Estrín, and Sara E. Bari Departamento de Química Inorgánica, Analítica y Química Física/ INQUIMAE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina. sbieza@qi.fcen.uba.ar     INTRODUCTION The protein environment of heme in hemeproteins is the maximum authority in the modulation of binding affinity and reactivity; a simple prove for this statement is the well-known fact that hemin IX (FeIII protoporphyrin IX, or hemin b) is the cofactor of different proteins with the most varied functions. Model systems and mutants have proved to provide a versatile and worthy experimental approach for studying the molecular determinants of the protein role. Systems of diverse structural complexities have been devised,1 with examples consisting of a heme moiety between two helical peptides,2 the covalent linked heme to a peptide chain (e.g. microperoxidase MP11),3 or the picket-fence porphyrins,4 among others. A minimalist model system for many hemeproteins needs to fulfil two requirements: 1) the fifth coordination position of the heme occupied by an imidazole-based ligand, and 2) a hydrophobic environment. It has been demonstrated that these requirements can be attained by dissolving hemin IX in a detergent solution over its critical micellar phase.5,6 We present our on-going studies on the use of sodium dodecyl sulphate (SDS) micelles for the evaluation of hemin IX reactivity towards inorganic sulphides, in the presence of imidazole-based fifth ligands. The hypothesis is that the imidazole-based fifth ligands can modulate the binding and/or reactivity of the Fe(III) towards hydrogen sulfide (H2S), devoid of distal counterparts. The question was raised on the current interest on the structural basis of the reactivity of endogenous H2S towards hemeproteins. This emerging issue is in the interest of physiologists, biochemists and chemists due to the role of H2S as second messenger.     EXPERIMENTAL METHODS Hemin IX dissolved in 0.1M NaOH (20 mL, conc. solution) was added to a solution of SDS (10mL, 1% in deaereated buffer PO43- 0.2M, pH 7.4). The concentration of SDS for monodispersed micelles was evaluated using transmission electron microscopy (Philips CM 200 (120 KV) and dynamic light scattering (90 Plus/ BI-MAS), from 1-10% SDS. The final solutions of hemin IX in SDS reached an absorbance ranging from 0.8-1.5. The addition of equivalent quantities of 2-methylimidazole was followed by the addition of H2S (g) and monitored by UV visible spectroscopy (HP-8453). The reactivity of the heme undecapeptide MP11 (buffer PO43-, pH 7.4), towards H2S was also assayed in SDS solutions. Control spectra for the Fe(II) species were obtained with sodium dithionite. H2S was prepared from Na2S.9H2O and phosphoric acid using Schlenk glass equipment. The gas was transferred with gas-tight syringes. Reagents were from Sigma-Aldrich Co.   RESULTS AND DISCUSSION The microscopy techniques suggest that the obtention of monodispersed SDS/hemin IX micelles was satisfactory accomplished for 1% SDS solutions. The absorption spectra of the visible and Soret regions of hemin IX reveal that aggregation is prevented in the presence of SDS (Figure 1).5 The addition of 2-methylimidazole (or 1,2-dimethylimidazole) show the characteristic spectra of a pentacoordinated species (Figure 1).6 The addition of H2S to hemin IX/2-methylimidazole in SDS (Figure 1) promptly induces a hypsochromic shift in the Soret band (l=384 nm), which is followed by the formation of a new complex (l max= 394nm). Significantly, this species is similarly attained in the absence of the base but, while in this condition the complex decays within an hour, the presence of the 2-dimethylimidazole maintains the same concentration of the complex after 24h. Figure 2. Reactivity of hemin IX towards H2S in SDS 1% solutions in the presence of 2-methylimidazole.   A preliminar analysis of the results suggests that the base does not induce a differential reactivity in the detergent solutions, but that the stability of a new complex is greatly enhanced in the presence of the base. The presence of the imidazole derivative yields a very stable complex, which deserves further characterization. These results are in line with our current research on the reactivity of the N- acetyl derivative of MP11, a heme undecapeptide that retains the proximal histidine and has no distal interactions.  The reaction of FeIIINacMP11 with sulfide species reveals that the proximal histidine allows stabilization of a sulfide ligand, devoid of distal counterparts.7       Figure 3. Reactivity of MP11 towards H2S in SDS 1% solutions.   To further support our concern on the proximal stabilization of the binding of sulfides to heme models, we assayed the monomeric MP11 in SDS solutions,8 after the addition of H2S, which depicts a similar spectrum to the one obtained for the non-aggregating N-acetyl MP11 in buffered solutions. These results suggest that the role of the proximal histidine in the stabilization of a (FeIII-sulfide) complex is likewise exerted in the aqueous solution or in the hydrophobic environment of the SDS micelles.   CONCLUSION The use of SDS micelles provides a simple procedure for the obtention of monomeric hemins. Coordination to different imidazole based ligands provides penta or hexacoordinated complexes, which are representative model compounds for reactivity studies. Concerning our studies of heme models towards inorganic sulfides, he present experiments reveal two aspects: 1) 2-methylimidazole can stabilize the formation of an hemin IX-sulfur complex, that deserves characterization, and 2) that  MP11 forms and stabilizes the same complex in aqueous solutions or in the hydrophobic SDS micelles, suggesting that the neutral H2S might be involved in the reactivity.   REFERENCES 1. Woggon, W. Acc. Chem. Res. (2005), 38:127-36. 2. Cordova, J.M. et al. J. Am. Chem. Soc. (2007), 129:512-518. (and refs. therein) 3. Marques, H. Dalton Trans (2007), 39:4361-4484. 4. Collman, J.P. et al. J. Am. Chem. Soc. (1975), 97:1427?1439. 5. Simplicio, J. Biochemistry (1975),11:2525-2534 6. Boffi, A. et al. Biophys. J. (1999), 77:1143-1149. 7. Bari, S.E. et al. Abstracts/ Nitric Oxide (2013) 31, S35. 8. Mazumdar, S. et al. Inorg. Chem. (1991), 30:700-705.   ACKNOWLEDGMENTS University of Buenos Aires, ANPCyT (PICT 2011-1266) and CONICET are acknowledged for financial support. Dr. Leonardo Boechi is acknowledged for introducing this approach in our research and for helpful discussions.