Analysis of the kinetic barriers for ligand binding to sperm whale myoglobin using site-directed mutagenesis and laser photolysis techniques

T. E. Carver, R. J. Rohlfs, J. S. Olson, Q. H. Gibson, R. S. Blackmore, B. A. Springer, S. G. Sligar

Research output: Contribution to journalArticlepeer-review

Abstract

Time courses for NO, O2, CO, methyl and ethyl isocyanide rebinding to native and mutant sperm whale myoglobins were measured at 20°C following 17-ns and 35-ps laser excitation pulses. His64(E7) was replaced with Gly, Val, Leu, Phe, and Gln, and Val68(E11) was replaced with Ala, Ile, and Phe. For both NO and O2, the effective picosecond quantum yield of unliganded geminate intermediates was roughly 0.2 and independent of the amino acids at positions 64 and 68. Geminate recombination of NO was very rapid; 90% rebinding occurred within 0.5-1.0 ns for all of the myoglobins examined; and except for the Gly64 and Ile68 mutants, the fitted recombination rate parameters were little influenced by the size and polarity of the amino acid at position 64 and the size of the residue at position 68. The rates of NO recombination and ligand movement away from the iron atom in the Gly64 mutant increased 3-4-fold relative to native myoglobin. For Ile68 myoglobin, the first geminate rate constant for NO rebinding decreased ~ 6-fold, from 2.3 x 1010 s-1 for native myoglobin to 3.8 x 109 s-1 for the mutant. No picosecond rebinding processes were observed for O2, CO, and isocyanide rebinding to native and mutant myoglobins; all of the observed geminate rate constants were ≤ 3 x 108 s-1. The rebinding time courses for these ligands were analyzed in terms of a two-step consecutive reaction scheme, with an outer kinetic barrier representing ligand movement into and out of the protein and an inner barrier representing binding to the heme iron atom by ligand occupying the distal portion of the heme pocket. Substitution of apolar amino acids for His64 decreased the absolute free energies of the outer and inner kinetic barriers and the well for non-covalently bound O2 and CO by 1 to 1.5 kcal/mol, regardless of size. In contrast, the His64 to Gln mutation caused little change in the barrier heights for all ligands, showing that the polar nature of His64 inhibits both the bimolecular rate of ligand entry into myoglobin and the unimolecular rate of binding to the iron atom from within the protein. Increasing the size of the position 68(E11) residue in the series Ala to Val (native) to Ile caused little change in the rate of O2 migration into myoglobin or the equilibrium constant for noncovalent binding but did decrease the unimolecular rate for iron-O2 bound formation. Decreases in the equilibrium constants for non-covalent methyl and ethyl isocyanide binding were observed for the same series of mutants, but again the largest effect was an increase in the height of the inner kinetic barrier when Ile was substituted for Val68. These results show that the isopropyl side chain of Vala68 comprises a portion of the inner steric barrier for iron-ligand bond formation. The Val68 to Phe mutation had little effect on the inner kinetic barrier and final equilibrium bound state. However, the affinities of ligands for the non-covalent binding site in the Phe68 mutant decreased 3-30-fold compared with native myoglobin, and the kinetic barrier for escape and entry into the mutant protein increased by 1-2 kcal/mol. The first result is due to decreasing the size of the distal cavity in the mutant protein. The effect of the Val68 to Phe substitution on the outer kinetic barrier may be due to inhibition of motions of the E helix that are required to open a channel between Val68 and His64 or to direct blockage of an alternative pathway for ligand entry.

Original languageEnglish (US)
Pages (from-to)20007-20020
Number of pages14
JournalJournal of Biological Chemistry
Volume265
Issue number32
StatePublished - 1990

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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