All globins consist of eight $\alpha$ helices labelled A through H and connected by loop regions, except mammalian $\alpha$ globins which lack a D helix. Myoglobin is used as a simple model for the study of the hemoglobin subunits to understand the stereochemical mechanisms that regulate function and stability in the globins. The roles of Phe43 (CD1), the D helix (residues 51-55), and Met55 (D5) in modulating the function and stability of sperm whale myoglobin were investigated by site directed mutagenesis. In myoglobin, Phe43$\to$Val, Leu, Ile, and Trp substitutions were constructed; the D helix was deleted and replaced with Ala$\sp{51-55}$ and Ala$\sp{51-54}$Met$\sp{55}$; and Met55(D5)$\to$Arg, Gly, Ala, Leu, Phe, and Trp substitutions were constructed. The role of the D helix (50-54) in $\beta$ subunits of human hemoglobin and the evolutionary significance of its absence from chordate human hemoglobin $\alpha$ subunits were examined by deleting residues 50-55 from $\beta$ subunits, called $\beta$ ($-$D), and inserting those same residues into $\alpha$ subunits, called $\alpha$ (+D). Finally, $\alpha$ His45 (CE3)$\to$Arg and $\beta$ Ser44 (CD3)$\to$Arg, Lys, Arg substitutions were constructed to increase hemin retention in recombinant human hemoglobin subunits.
The size, hydrophobicity, and aromaticity of Phe at CD1 are required to regulate ligand binding, inhibit autooxidation, and retain hemin. The D helix appears to stabilize the CD corner and inhibit rapid hemin loss. A large apolar residue at D5 immobilizes the D helix, indirectly slowing hemin loss. Removal of the D helix from $\beta$ subunits also destabilizes bound hemin in both hemoglobin subunits. The absence of a D helix in chordate $\alpha$ subunits appears to be compensated by contacts with the adjacent $\beta$ subunits across the $\alpha$1$\beta$1 interface. This interface appears to stabilize the CE corner to inhibit hemin loss. A large apolar residue is required at positions CD1 and D5 to stabilize the heme binding pocket in apomyoglobin.
Arg45 (CD3) stabilizes bound hemin in myoglobin by forming a salt bridge with the heme-6-propionate. His44 (CE3) in $\alpha$ subunits forms a similar salt bridge, whereas Ser45 (CD3) does not in $\beta$ subunits. When Ser44 (CD3) is replaced with His a favorable interaction with the heme-6 propionate appears to occur, stabilizing the $\beta$ globin-heme complex. Since this mutation has little effect on ligand binding and autooxidation, it appears to be a good secondary mutation for stabilizing a cell-free Hb-based blood substitute.
| Identifer | oai:union.ndltd.org:RICE/oai:scholarship.rice.edu:1911/16913 |
| Date | January 1995 |
| Creators | Whitaker, Timothy Lee |
| Source Sets | Rice University |
| Language | English |
| Detected Language | English |
| Type | Thesis, Text |
| Format | application/pdf |
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