Association and dissociation rate constants were measured for O2, CO, and alkyl isocyanide binding to a set of genetically engineered sperm whale myoglobins with site-specific mutations at residue 64 (the E7 helical position). Native His was replaced by Gly, Val, Leu, Met, Phe, Gln, Arg, and Asp using the synthetic gene and expression system developed by Springer and Sligar (Springer, B.A., and Sligar, S.G. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8961-8965). The His64 → Gly substitution produced a sterically unhindered myoglobin that exhibited ligand binding parameters similar to those of chelated protoheme suspended in soap micelles. The order of the association rate constants for isocyanide binding to the mutant myoglobins was Gly64 (~ 107 M-1 s-1) >> Val64 ~ Leu64 (~ 106 M-1 s-1) > Met64 > Phe64 ~ His64 ~ Gln64 (105 - 103 M-1 s-1) and indicates that the barrier to isocyanide entry into the distal pocket is primarily steric in nature. The bimolecular rates of methyl, ethyl, n-propyl, and n-butyl isocyanide binding to the His64 → Arg and His64 → Asp mutants were abnormally high (1-5 x 106 M-1 s-a1), suggesting that Arg64 and Asp64 adopt conformations with the charged side chains pointing out toward the solvent creating a less hindered pathway for ligand binding. In contrast to the isocyanide data, the association rate constants for O2 and CO binding exhibited little dependence on the size of the E7 side chain. The values for all the mutants except His64 → Gln approached or were larger than those for chelated model heme (i.e. ~ 1 x 108 M-1 s-1 for O2 and ~ 1 x 107 M-1 s-1 for CO), whereas the corresponding rate parameters for myoglobin containing either Gln64 or His64 were 5- to 10-fold smaller. This result suggests that a major kinetic barrier for O2 and CO binding to native myoglobin may involve disruption of polar interactions between His64 and water molecules found in the distal pocket of deoxymyoglobin. Finally, the rate and equilibrium parameters for O2 and CO binding to the His64 → Gln, His64 → Val, and His64 → Leu mutants were compared to those reported previously for Asian elephant myoglobin (Gln-E7), Aplysia limacina myoglobin (Val-E7), and monomeric Hb II from Glycera dibranchiata (Leu-E7).
|Original language||English (US)|
|Number of pages||9|
|Journal||Journal of Biological Chemistry|
|State||Published - 1990|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology