Quantum yields and the mechanism of ligand binding to heme proteins
Rohlfs, Ronald James
Doctor of Philosophy
Ligand binding to several naturally occurring and engineered myoglobins and hemoglobins was examined. Overall and germinate rate constants and quantum yields were determined at pH 7, 20$\sp\circ$C for O$\sb2$, NO, CO, and a set of n-series and $\alpha$-substituted alkyl isocyanide complexes of sperm whale myoglobin, isolated $\alpha$ and $\beta$ hemoglobin chains, soybean leghemoglobin, and monomeric hemoglobin component II from Glycera dibranchiata using a 30 ns dye laser pulse and a 0.5 ms xenon flash. Overall rates and quantum yields for several sperm whale myoglobin complexes were compared to parameters obtained for site directed mutants of this protein, in which His-E7 was replaced by one of several different amino acids. Results were analyzed using a linear three step reaction scheme, HbX$\rightleftharpoons$B$\rightleftharpoons$C$\rightleftharpoons$H $+$ X, where X is ligand free in solution, H and HX are unliganded and liganded heme protein respectively, B represents a geminate state in which the ligand is in the distal pocket but not covalently bound to the iron atom, and C is a state in which the ligand is still embedded in the protein but further away from the heme group. The results indicate that low overall quantum yields are due to rapid rates of geminate recombination relative to rates of ligand escape to solvent rather than a low intrinsic photophysical yield. For all CO complexes, Q is large and the rate limiting step for association is iron-ligand bond formation and dissociation is rate limited by thermal bond disruption. For O$\sb2$, NO, and most of the isonitrile complexes, Q is small ($\leq$0.2) indicating the association rate is approximately equal to the rate of ligand migration to state B, and dissociation is limited by escape from the protein. Studies of the site directed mutants of Mb show that the E7 residue acts as a steric barrier for isonitriles. In contrast, the polarity of the E7 side chain is more important in determining the rate and equilibrium constants for O$\sb2$ and CO binding. The dominant factor for the stabilization of the native MbO$\sb2$ complex is the hydrogen bond between bound O$\sb2$ and the N$\varepsilon$ proton of His-E7. The largest kinetic barrier to O$\sb2$ and CO binding appears to be displacement of water found in the distal pocket of native deoxymyoglobin.