Kinetic barriers to ligand binding in myoglobin

[摘要] Picosecond, nanosecond, and bimolecular reactions of ligands with myoglobins containing distal pocket mutations at positions 29, 45, 64, and 68 were examined at 20$spcirc$C, in 0.1 M potassium phosphate pH 7.0. The Val$sp{68}{o}$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$sp{29}{o}$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$sp{29}{o}$Phe mutation caused the distal pocket to become more compartmentalized, enhancing picosecond recombination and hindering nanosecond recombination. The Leu$sp{29}{o}$Val and Leu$sp{29}{o}$Ala substitutions caused picosecond and nanosecond intermediates to become less distinct. The Val$sp{68}{o}$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$sp{68}{o}$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 $imes$ 10$sp7$s$sp{-1}$, regardless of mutations in the distal pocket.

[发布机构] Rice University

[效力级别] Biophysics