Spectroscopic studies of myoglobin mutants and the kinetics of water entry into the heme pocket.

摘要

Previous studies showed that time resolved optical absorption (TROA) can measure the occupancy of non(heme)coordinated water in the distal pocket of myoglobin with high fidelity. Internal water molecules are important to protein structure and function, but positional disorder and low occupancies can obscure their detection by X-ray and NMR structural methods. Here we monitored the kinetics of CO photodissociation and heme hydration in sperm whale myoglobin as a function of amino acid substitution of three highly conserved residues in close proximity to the heme pocket: L29, H64 and V68. These studies disentangled the direct effects of residue size and hydrophobicity on heme pocket hydration. The mutants presented in this study are myoglobin mutants L29W, L29F/V68L, H64L/V68N, L29F, V68L, L29F/H64L in addition to WTMb. The information obtained from mutants in this study, together with the information from previous studies, can be used to determine the dominant mechanism by which active site mutations affect the bimolecular rate constant for CO binding. Three conclusions reached from this study. First, TROA spectral assay can be reliably used to detect both ordered and disordered water in the heme pocket. Second, the presence of disordered water is facilitated by side chains of not only the His 64 residue but also other residues in the heme pocket such as L29 and V68. Size, flexibility and electrostatic characteristics of L29 and V68 mutant side chains can affect water's presence in the pocket. Third, the data from this study, in combination with data from previous studies indicate that water in the heme pocket affects CO ligand binding kinetics more than any other factor, such as the size of heme pocket residues or geminate recombination. The increase of water occupancy in the heme pocket results in a decrease of the rate of CO recombination.