Spectroscopic studies of ligand migration in myoglobin

Abstract

Myoglobin (Mb) is a globular protein that binds small ligands (O2, CO, NO). It was realized early on that the x-ray structure lacked an access pathway for the ligand to the active site; consequently, protein fluctuations that open transient channels must be essential for its function. The aim of this thesis research was to elucidate the dynamic aspects of the ligand binding reaction, especially the role of internal protein cavities. The key strategy was to remove or strongly modify the cavities by simple competitive binding of xenon, and more elegantly using modern tools of protein engineering. The ligand bond in myoglobin is photolabile and can be dissociated by a short laser pulse. Fourier transform infrared (FTIR) spectroscopy was employed to follow CO migration through the protein. The spectroscopic work was done over a wide temperature range (3-300 K). For the wild type MbCO and a whole family of single point mutants, a number of photoproduct states have been identified that are associated with ligands trapped in different cavities in the protein. Local structural changes were shown to have significant effects on the accessibility of a particular site for CO. To connect the low-temperature spectroscopic data with physiological ligand binding, room temperature flash photolysis data were collected on MbCO mutant samples. A thermodynamic model of ligand binding was developed that considers the internal cavities as transient ligand storage sites. In this model, both bond formation and ligand entry and exit are slow processes compared with the equilibration among the internal sites. By fluctuating among the internal sites, the photolyzed ligands can take advantage of protein fluctuations and escape. The exact route(s) for ligand entry and exit remained obscure.