Román Morales, Elddie M.

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    Global and local structural analysis of the sulfheme complex: A role in the decrease of myoglobin functionality
    (2016) Román Morales, Elddie M.; López Garriga, Juan; College of Arts and Sciences - Sciences; Vega Olivencia, Carmen A.; Hernández Rivera, Samuel P.; Rivera Portalatin, Nilka; Department of Chemistry; Almodovar, Jorge
    When the human body is exposed to high concentration of hydrogen sulfide (H2S, recently identified as a signaling gas), a rare type of anemia called sulfhemoglobinemia is developed. This condition is triggered by the increase of the physiological concentration of sulfheme complex. Proteins bearing a His distal residue with an adequate orientation, as in Myoglobin (Mb) and Hemoglobin (Hb), form this complex. The sulfur atom is incorporated across the β-β double bond of the pyrrole “B”, reducing the protein‟s oxygen (O2) affinity. This change in affinity is attributed to the heme group distortion and the electron withdrawing forces induce by the sulfur atom. However, other aspects may also contribute to the decrease in protein affinity, e.g. conformational change and final bound heme Fe-ligand. Our intention was to analyze the contribution of these global and local structural features with those previously reported and relate them to the changes in protein functionality. Therefore, this research was directed to further comprehend the reactivity and action mode of H2S in the human body, specifically Mb. In this project we: (1) evaluated the possible significant changes in protein conformation that may affect the protein-O2 interaction, and (2) identified the SulfmyoglobinLigand (SMb-L) complexes that naturally take place as products during the SMb complex formation, and hence, serve as physiological competitors for the binding of O2 to the heme-Fe. To answer these inquiries, Small angle X-ray scattering (SAXS) and Electron Paramagnetic Resonance (EPR) were used to analyze the SMb complex, along with control protein HbI from Lucina pectinata that does not form the sulfheme complex. The EPR data showed that the possible final products of the SMb complex formation are H2O-SMb (2.49, 2.26, and 1.84 g values), O2-SMb (loss of signal), ferrous (loss of signal) and ferric H2S-SMb (2.36, 2.26 and 1.91 g values). In order for O2 to bind to the sulfheme-Fe, it will have to compete with H2O and H2S ligands, in addition to the ferric heme-Fe oxidation state, which contributes to the loss of O2-protein interaction. Finally, the SAXS results showed that SMb formation induces a change in the protein conformation; where its envelope forms a very small cleft and the protein is more flexible, less rigid and compact. Theoretical scattering curve of SMb crystal structure suggest that the global conformational change and internal structural fluctuations are hampered by the crystal packing forces; thus, limiting the range of conformational motion accessible to the protein. Based on the direct relationship between Mb‟s structural conformation and its functionality, we suggest that the conformational change observed upon SMb formation plays a role and contribute to the protein decrease in O2 affinity and, therefore, on its functionality.
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    Effect of the Histidine E7 amino acid in the sulfheme formation of the hemoglobin I from Lucina Pectinata
    (2008) Román Morales, Elddie M.; López Garriga, Juan; College of Arts and Sciences - Sciences; Vera, Marisol; Ríos, Robert; Department of Chemistry; Aponte, Nilda E.
    Sulfhemoglobin is a non-functional derivative of hemoglobin known to be produced by exposure to sulfa drugs, air pollution and others. It is formed by the reaction between H2O2, H2S and the heme group in the presence of oxygen with the corresponding specific addition of sulfur to the pyrrole B of the heme. It is characteristic spectroscopic absorption is observed around 620nm. The intermediates in the reaction are the heme Fe IV=O ferryl species, known as Compound I and II. This complex is of immense importance in the medical community because it is one of the main forms of nonfunctional hemoglobin that produces anemia. This derivative is commonly observed in most hemeproteins with the exception of HbI, HbII, and HbIII, hemeproteins, from the clam Lucina pectinata. Studies performed over the years with these Hb’s and H2S in the presence of O2 showed no formation of the sulfheme complex. Therefore, it is important to determine the chemical structure of the protein responsible for the formation of sulfhemoglobin. UV-Vis spectroscopic analyses were made on human Hb, HbI, HbII/HbIII, HbI PheE11Val, HbI GlnE7His, HbI PheB10His, HbI PheB10Val protein samples upon reaction with hydrogen sulfide and hydrogen peroxide, using horse heart myoglobin (Mb) as the control. Surprisingly, the only HbI mutant to form the sulfheme complex was HbI GlnE7His variant, evidenced by the formation of the 626nm band. These studies revealed that only heme proteins with histidine in the specific E7 position form the unique sulfheme derivative. However, the sulfMb(O2) final product may co-exist with a six coordinated high spin species while the SHbI GlnE7His final product could coexist with a low spin oxy complex, as show by Optical and 1 H NMR Spectra. In addition, the results indicated that neither compound I nor compound II play a significant role in the formation of the sulfheme derivative since HbI and HbII/HbIII, which form stable compound I and compound II, respectively, did not form the sulf species. Thus, it is suggested that if any ferryl species is involved in the formation of sulfheme complex, it should be the one where His in the E7 position interacts directly with the bound hydrogen peroxide, as in the hydroperoxy complex.