Protein engineering studies of myoglobin
Creators: name: Maurus, Robert
Publisher: name: University of British Columbia
Year: 2009

description: The goals of the work described in this thesis were to gain further insight into structure-function relationships in the oxygen binding protein myoglobin, alter the structure of this protein to enhance a low level latent peroxidase function, and to add a new functionality in the form of a manganese binding site. An initial step in these studies was the elucidation of the high resolution structure of recombinant horse heart myoglobin to confirm that this protein is properly expressed and folded in a manner comparable to horse heart myoglobin obtained from natural sources. The additional variant myoglobins that were studied were organized into three groups. The first was constructed to probe aspects of the role of the distal heme pocket residue His64. Replacement of this histidine with threonine generated a five coordinate heme iron atom in the ferric state, whereas substitution with tyrosine yielded a six coordinate heme iron atom. The lack of peroxidase activity in the His64Thr variant protein appears to be a direct result of an inability to participate effectively in proton transfer during peroxide heterolysis. For the His64Tyr variant protein, the difficulty of dissociating the distal tyrosine ligand from the heme iron center appears to prevent hydrogen peroxide binding to the heme and thus limit peroxidase activity. Additional experiments were designed to facilitate the interpretation and understanding of the structural consequences of ligand binding to myoglobin. In particular, structural characterizations were completed of azide - heme ligation complexes with wild-type recombinant horse heart myoglobin and the His64Thr variant protein. These studies established that there are no large structural rearrangements required for the formation of an azide complex with either protein. However, substitution of His64 with threonine did show two unexpected effects in terms of azide complex formation. These included the observation of two bound azide conformations and on the proximal side of the heme pocket, the presence of a shorter hydrogen bond between Ser92 and the proximal heme iron ligand His93. The second group of variant proteins studied was prepared to probe the influence of amino acid substitutions in the heme binding pocket. These included replacements at the distal heme pocket residues Leu29 and Val67, and at Leu 104 which is located on the proximal side of the heme. These studies showed that substitution of Leu29 for a tyrosine or a lysine can be accommodated in the heme pocket of myoglobin. The peroxidase activity of both variant proteins is higher than that of wild-type myoglobin. This increase in activity appears to be correlated primarily with an increase in the polarity of the distal heme pocket. Surprisingly, the observed increase in heme reduction potential for the Val67Arg variant protein was also accompanied by an increase in peroxidase activity to approximately 8 times that of wild-type myoglobin. The Leul04Asn variant protein also showed an approximate 3-fold increase in activity towards hydrogen peroxide. A third group of variant proteins was designed to build a functional manganese binding site into horse heart myoglobin. Structural studies of the Lys45Glu and Lys45Glu/Lys63Glu variant proteins were conducted and indicate manganese binding occurs at the solvent exposed edge of the heme with ligands being formed by Glu45 and the heme D-propionate group. These residues constitute a site that is comparable to the one found later in structural studies of a manganese peroxidase by other workers. These myoglobin variants show ~ 3-fold enhancement in the rate of oxidation of Mn(II) to Mn(III) in the presence of hydrogen peroxide. Additional structural studies using Cd(II) ions were also conducted, and in this case two binding sites were located. Overall, the present work demonstrates that amino acid substitutions can influence the latent peroxidase activity of myoglobin and that it is possible to construct a Mn(II) ion binding site at an appropriate position on the surface of myoglobin to promote detectable manganese peroxidase activity.