Ramos Santana, Brenda J.

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    NMR structural determinants of lucina pectinata hemoglobin I active center moieties
    (2013) Ramos Santana, Brenda J.; López Garriga, Juan; College of Arts and Sciences - Sciences; Prieto, José; Meléndez, Enrique; Cadilla, Carmen; Department of Chemistry; Navarro, Ana J.
    The clam Lucina pectinata Hemoglobin I (HbI) is a monomeric protein with a prosthetic group, known as heme group, in which the iron atom can either be in the ferrous (Fe(II)) or the ferric (Fe(III)) oxidation state. This globin protein is sulfide reactive hemoglobin in its ferric state that binds and transports H2S to sulfide-oxidizing chemoautotrophic bacteria to maintain a symbiotic relationship. It was discovered that H2S binding to HbI has a biological function protecting the mollusk from H2S toxicity as an adaptative process of the invertebrate hemoglobin I from L. pectinata. The distal pocket of HbI is characterized by the presence of a GlnE7 and a PheB10. Site-directed mutant of the gene encoding wild-type HbI were made that changed Phe to Tyr at B10 position. The recombinant (rHbI) and rHbI PheB10Tyr mutant proteins were expressed in Escherichia coli and purified to homogeneity. 1H-NMR studies were conducted on rHbI and rHbI PheB10Tyr mutant to elucidate the structural-functional properties of HbI. The effects of this mutation were evaluated on the ferric cyanide derivative environment to characterize the structural properties of the rHbI and rHbI PheB10Tyr mutant. The results obtained show chemical shifts of TyrB10 OHn at 31.00 ppm, GlnE7 NE1H/NE2H at 10.66/-3.27 ppm, and PheE11 CH at 11.75 ppm, indicating the presence of a crowded, collapsed, and constrained distal pocket. The strong dipolar contacts and inter-residues crosspeaks between GlnE7/6-heme propionate group, GlnE7/TyrB10 and TyrB10/CNresults suggested that a hydrogen bonding network loop between GlnE7, TyrB10, 6-propionate group, and the heme ligand contribute significantly to the modulation of the heme iron electron density as well as the ligand stabilization mechanism. Therefore, the network loop presented here support the fact that the electron withdrawing character of the hydrogen bonding is controlled by the interaction of the propionates and the nearby electronic environments contributing to the modulation of the heme electron density state. Thus, we hypothesize that in hemeproteins with similar electrostatic environment the flexibility of the heme-6-propionate promotes a hydrogen bonding network loop between the 6-propionate, the heme ligand and nearby amino acids, tailoring in this way the electron density in the heme-ligand moiety. Also, a NMR structural model was used to reveal a magnetic field mapping around the heme oxidation-reduction state in presence of H2S derivative.