Effect of heme pocket modifications on the structure and activity of myoglobin
Creators: name: Burk, David Logan
Publisher: name: University of British Columbia
Year: 2009

description: The experiments described in this thesis were conducted as part of an effort to investigate the possibility of enhancing the latent peroxidase activity of horse heart myoglobin. This investigation could then form a basis for the development of a protein to assist in the delignification of wood pulp. The experiments performed centred on the modeling of appropriate variant proteins, the determination of the three dimensional atomic structures of eight variant myoglobin structures by X-ray diffraction methods, the systematic analysis of these structures and their comparison with the structure of wild-type horse heart myoglobin. In addition, assays of the peroxidase activity of variant proteins were performed and these results were correlated with structural data. The eight variant proteins studied can be organized into three principal categories. The first group focussed on the proximal heme iron ligand (His93) of myoglobin and the adjacent Ser92 residue, which forms a hydrogen bond interaction with His93. Replacement of the proximal ligand with tyrosine (H93Y) yielded a five-coordinate heme iron centre with tyrosine ligation, a large shift in a nearby helix, and a peroxidase activity which was approximately half that of wild-type myoglobin. The second protein analyzed (S92D) was hypothesized to enhance peroxidase activity by forming a strong hydrogen bond with the proximal ligand. The structure revealed, however, that this substitution could not be accommodated in this manner. Despite this, the peroxidase activity of the S92D protein was 50% higher than that of wild-type myoglobin. In addition, the structure of this variant protein suggests a role for Ser92 in the maintenance of the orientation of proximal heme pocket components and in modulating the chemical properties of the heme. The second category of variant structures were those prepared to probe the influence of amino acid substitutions at the outer edge of the proximal heme pocket. These studies show that the introduction of phenylalanine or tyrosine residues at position 97 does not result in proteins with significantly increased peroxidase activity. In contrast to these aromatic substitutions, a branched aliphatic leucine substitution (H97L) was found to increase peroxidase activity to five times that of wild-type myoglobin. Hydrogen bonding interactions involving residue 97 were shown to be important in maintaining both the orientation of the heme pyrrole A propionate and the geometry of the proximal heme ligand. The final group of proteins were studied to probe the effect of K45E and K45E/K63E/S92A substitutions on the structure and activity of the S92D and H97L proteins. The K45E and K45E/K63E/S92A substitutions form a putative metal binding site required for manganese peroxidase activity in myoglobin. Structural studies show that the substitutions involved in the construction of the metal binding site do not significantly disrupt the structure of the heme pocket. The observed increase in peroxidase activity associated with variant proteins containing additional substitutions related to the construction of a metal binding site is likely the result of increased accessibility of the heme to substrate, although other factors may also be involved.