The Effect of Distal Heme Pocket Mutations on the Water Accessible Areas in Myoglobin

Abstract

Internal water molecules are important to protein structure and function. A non-coordinated water molecule in the distal pocket of a myoglobin has been shown to be the dominate factor in controlling the binding of CO to the heme active site. We previously developed a method to experimentally measure the entry of internal water into the distal pockets of Mb mutants after photodissociation of CO. In order to better understand what factors control the occupancy of this disordered water in the protein we compared the occupancy with the size of the mutated residue and hydrophobicity. We see little correlation between residue size and water occupancy and a good correlation between water occupancy and hydrophobicity. In order to better understand what factors contribute to internal water occupancy, we further examined how cavity volume and the dynamic behavior of the distal histidine influence water occupancy. Using a computational approach, we calculated the internal volumes of myoglobin cavities for various mutants. We further characterized these cavities by investigating the dynamic behavior of the H64 residue using molecular dynamics. The data show high flexibility of the H64 in the wild type protein suggesting a mechanism by which water is allowed access to the distal cavity. However, in the distal pocket mutants, the H64 can adopt a more stable conformation thereby reducing water access to the cavity. These findings suggest that the flexibility of the distal histidine plays a key role in influencing water access to the distal cavity and the binding affinity for gaseous ligands. In addition, the long range molecular dynamics was used to assess stability of the cavity bound water for the various mutants. The obtained data showed correlation between hydrophobicity and the water residence time in the cavity.