dc.contributor.advisor Olson, John S. Carver, Theodore Edward, Jr 2009-06-04T00:41:18Z 2009-06-04T00:41:18Z 1993 https://hdl.handle.net/1911/16606 Picosecond, nanosecond, and bimolecular reactions of ligands with myoglobins containing distal pocket mutations at positions 29, 45, 64, and 68 were examined at 20$\sp\circ$C, in 0.1 M potassium phosphate pH 7.0. The Val$\sp{68}{\to}$Ile mutation hindered intramolecular iron-ligand bond formation, slowing recombination of NO on picosecond time scales and O$\sb2$ on nanosecond time scales. Picosecond NO recombination was enhanced by increasing the size of residue 29. The rates for the major picosecond rebinding phase were 1.8, 2.5, 29, and $\ge$100 ns$\sp{-1}$ for Ala$\sp{29}$, Val$\sp{29}$, Leu$\sp{29}$(native), and Phe$\sp{29}$ myoglobin. In contrast to this trend, the Leu$\sp{29}{\to}$Phe mutation caused a 10-fold decrease in the rate of nanosecond NO recombination. These effects were interpreted in terms of a model for picosecond and nanosecond ligands based on diffusion of ligands within the protein matrix. On picosecond time scales, the photodissociated ligand appears to reside in a space bordered by His$\sp{64}$ and Leu$\sp{29}$ close to the iron atom. On nanosecond time scales, unbound ligands have diffused farther away into a space adjacent to Ile$\sp{107}$. The Leu$\sp{29}{\to}$Phe mutation caused the distal pocket to become more compartmentalized, enhancing picosecond recombination and hindering nanosecond recombination. The Leu$\sp{29}{\to}$Val and Leu$\sp{29}{\to}$Ala substitutions caused picosecond and nanosecond intermediates to become less distinct. The Val$\sp{68}{\to}$Phe mutation enhanced nanosecond O$\sb2$ recombination by reducing the volume available to the unbound ligand. Thus, distal pocket structure plays a key role in the internal kinetic barriers to ligand recombination. Replacement of His$\sp{64}$ with apolar residues facilitated ligand entry into the protein, due to loss of the distal water molecule in deoxymyoglobin. The Val$\sp{68}{\to}$Thr mutation produced the opposite effect, hindering ligand entry by stabilizing the distal H$\sb2$O. No definite information was gained about pathways of ligand entry and escape, but there appears to be a global protein barrier to ligand exit that causes the rate of ligand escape to be $\sim$1 $\times$ 10$\sp7$s$\sp{-1}$, regardless of mutations in the distal pocket. 192 p. application/pdf eng BiochemistryBiophysics Kinetic barriers to ligand binding in myoglobin Thesis Text Chemistry Natural Sciences Rice University Doctoral Doctor of Philosophy Carver, Theodore Edward, Jr. "Kinetic barriers to ligand binding in myoglobin." (1993) Diss., Rice University. https://hdl.handle.net/1911/16606.
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