Molecular recognition by genetically engineered myoglobins
Creators: name: Hunter, Christie Lynne
Publisher: name: University of British Columbia
Year: 2009

description: Specific formation of non-covalently associated complexes between biomolecules is a phenomenon of fundamental importance in biology. The present study examines a variety of myoglobin variants that have been designed as models of such "molecular recognition" processes. Myoglobin (Mb) has been selected for this purpose because it is a small (17.6 kDa) protein with a non-covalently bound heme prosthetic group that can bind dioxygen and other ligands reversibly and because recombinant Mbs can be expressed efficiently in high yield. As summarized below, three types of molecular recognition processes have been considered: (a) the interaction of divalent metal ions with a metal binding site that has been introduced on the surface of the protein near the partially exposed heme edge, (b) the contribution of the hydrogen bonds formed between the heme propionate groups and protein residues to the stabilization of the interaction of heme with apoMb, and (c) the influence of amino acid residues in the active site of the protein on the ability of the heme iron to bind azide. As part of this work, relevant spectroscopic and electrochemical properties of the variants involved have also been considered. A binding site for metal ions was engineered on the surface of horse Mb near the heme 6- propionate, similar to the site present in the manganese peroxidase enzyme produced by the white-rot fungus, Phanearochaete chrysosporium. The equilibrium constant for Mn(II) binding to engineered site was determined to be 1.3 x 10⁴M⁻⁻1 (pH 7.0, 25 °C, 1=17.2 mM), and the affinity was shown to decrease at lower pH and higher ionic strength. A low level of oxidation of Mn(II) to Mn(III) in the presence of H₂O₂ was observed in the variants of Mb possessing enhanced metal binding. Other metal ions were shown to bind with varying affinities to this engineered site, Cd(II) &gt; Co(II) &gt; Cu(II) ≈ Mn(II). To study the importance of the hydrogen bonding interactions between the heme propionates and the protein matrix on heme binding dynamics and stability, a series of variants and derivatives of horse heart Mb and bovine liver cytochrome b₅ was constructed in which the amino acid residues that form hydrogen bonds with the heme propionate groups were systematically substituted. These hydrogen bonding interactions play a partial role in stabilizing the heme within the binding pocket as suggested by the observed decreased thermal stability, the increased rate constants for heme dissociation, and the lower activation energies for heme dissociation of the Mb and cytochrome b₅ variants. These hydrogen bonding interactions play a more significant role in the heme reorientation kinetics as indicated by the greatly increased rate constants for heme reorientation observed for the Mb variants. The correlation between the stability of protein-small molecule complexes in the gas phase and their stability in aqueous solution was evaluated using electrospray mass spectrometry through the investigation of the series of horse heart Mb and bovine liver cytochrome b₅ variants described above. A good correlation between the stability of heme binding in the gas phase and in solution was obtained which suggests the hydrogen bonding interactions present in solution are maintained in the gas phase under these mild conditions and that the protein is not grossly misfolded in the gas phase on the time scale of these measurements. Charge reversal mutations close to the heme pocket had significant effects on the properties of the distal heme binding pocket, specifically, the binding of the anionic azide ligand, the pKA of the distal water molecule and the reduction potential of the heme iron. Metal binding to the surface of the protein near the heme affects the properties of the distal heme binding pocket, exhibiting linkage to the binding affinity for anionic ligands and to the reduction potential of the heme iron.