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Structural and Biochemical Characterization of the Hemophore (HasAp) From Pseudomonas Aeruginosa
Alontaga, Aileen Yung
Alontaga, Aileen Yung
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Abstract
HasAp is a hemophore which is believed to "steal" heme from hemoglobin in the extracellular medium and deliver the heme to the cognate receptor, HasR for internalization. The mechanism of heme uptake and release mechanism of HasAp has not been understood. To gain insight on this mechanism, we have elucidated the electronic and coordination state of the heme active site, using a combination of bioanalytical techniques. The 13C-meso chemical shifts were primarily used as a straightforward probe for the coordination and electronic state of HasAp. The results showed that heme-iron is hexacoordinate and in equilibrium between high-spin (S = 5/2) and low-spin (S = ½). The proposed explanation this unique coordination and spin state is the breaking and forming of hydrogen-bond between the axial ligand Tyr75 and His83 which could modulate the heme uptake and release in HasAp. The heme transfer experiments reveal that HasAp can take heme only from hemoglobin if the heme-iron is in the ferric state. It can not take heme from met-myoglobin, oxyhemoglobin and HbCN. The weak aqua ligand that coordinates the ferric iron in met-Hb is essential to the heme transfer process since its substitution with a stronger ligand inhibits the heme transfer to HasAp. Moreover, the rate of heme transfer is relatively fast and it occurs in two steps. These observations imply that there appear to be a protein-protein interaction between HasAp and hemoglobin before HasAp take the heme from hemoglobin. Hence, the heme transfer event from met-Hb to apo-HasAp is a multi-step process. The secondary structure of holo-HasAp was determined using X-ray crystallography and NMR spectroscopy. The results showed that the last 21 amino acid residues which composed the tail of the full-length form are highly unstructured and disordered. There is no structural difference in both the holo full-length and truncated form of HasAp except for the tail. However, there is a conformational difference between the apo and holo forms of HasAp. The regions close to the heme active site are in different conformations in both forms which could suggests that the protein uses different bonding interaction to stabilize the heme. From the protein-protein interaction experiments the data shows that the same regions of the secondary structure in both holo full-length and truncated HasAp were perturbed upon hemoglobin binding. However, the residues in the truncated form are more perturbed which suggests that the truncated form is likely to be the biologically active form of HasAp.
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Date
2008-07-23
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University of Kansas
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Analytical chemistry