Nieves Marrero, Carlos A.

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    Crystallographic structures of hemoglobin II (HbII) from Lucina Pectinata in the 4–9 pH range
    (2011) Nieves Marrero, Carlos A.; López Garriga, Juan; College of Arts and Sciences - Sciences; Hernández Rivera, Samuel P.; García Ruiz, Juan M.; Cadilla Vázquez, Carmen L.; Department of Chemistry; Navarro Rodríguez, Ana J.
    Lucina pectinata’s ctenidia contains three hemeproteins: the hydrogen sulfide reactive hemoglobin I (HbI) and the oxygen transporting hemoglobins II and III (HbII and HbIII) that remain unaffected by the presence of H2S. The mechanism for ligand discrimination used by these hemeproteins, their function and ligand selection and stabilization remain unknown. The distal pocket composition of HbII contains TyrB10 and GlnE7 residues. They could contribute to the ligand stabilization with a hydrogen bond network between the ligand and the residues. Also, the heme group of HbII is buried, compared to HbI, within the protein matrix. The synergetic effects of HbII’s heme pocket size, in combination with the hydrogen bonding network and the orientation of the heme–oxygen complex, confer stability to this heme derivative. The pH could produce rearrangements in the heme pocket of the protein and also trigger changes on its native structure and functionality. In this work, crystals of oxy–HbII at different pHs were obtained and their x-ray diffraction collected using the synchrotron radiation facilities in Grenoble, France. The corresponding crystallographic structures were used to evaluate the structural changes induced by pH, particularly around the TyrB10 and GlnE7 residues. Oxy–HbII crystals were grown at different pH values by the capillary counterdiffusion (CCD) technique, using a two–step protocol. A mini screen (first step) was used to validate sodium formate as the best precipitating reagent to grow oxy–HbII crystals. The second step, a pH screen typically used for optimization, was used to produce the crystals in the pH range from 4 to 9. Very well faceted prismatic ruby–red crystals were obtained at all pH values. X-ray diffraction data sets were acquired using synchrotron radiation of wavelengths of 0.886 Å (at pH 5) and 0.908 Å (at pH 4, 8 and 9) to a maximum resolution of 3.30, 1.95, 1.85 and 2.00 Å for pH 4, 5, 8 and 9, respectively. All crystals obtained were isomorphous, belonging to the P42212 space group. The four crystallographic structures were solved and uploaded to the RCSB Protein Data Bank, each receiving the following PDB codes: 3PI4 for pH4, 3PI3 for pH5, 3PI2 for pH8 and 3PI1 for the pH9 structure. The crystallographic results suggest the pH effect is a possible driving force for a conformational change in the HbII protein structure, specifically in the vicinity of the distal region. The orientation of the peripheral vinyls and propionate groups was also affected by pH. Slight changes were observed in the rearrangement of the hydrogen bond network of the TyrB10 and GlnE7 with the oxygen molecule at the heme pocket of HbII at pH5, 8 and 9. The structure at pH4 presents a porphyrin ring deformation as consequence of the entrance of a water molecule to the distal site. This produced a metaquo–HbII complex as consequence of the heme iron oxidation (Fe+2 -> Fe+3). The overall structures for Lucina pectinata HbII evidenced slight variations in the secondary structure as a function of pH, but the integrity of the classical 3–over–3 protein globin fold remained unaffected. Finally, the crystallographic results in the particular oxy–heme TyrB10–GlnE7 moiety suggest a mechanism for oxygen dissociation from the heme group by the relevance of the pH effect at the heme pocket residues. Specifically, as the pH range decrease from 9 to 5, the Fe–O2 bond weakens as the bond length increase from 1.8 Å to 2.0 Å. This suggests that the conformation and amino acids protonation changes are a pathway for the release of the oxygen molecule in these systems. The result indeed confirms the predominant role of the TyrB10 in the stabilization and dissociation of the oxy–heme ligand complex in hemeproteins.