Kinetic and cooperative mechanisms of ligand binding to hemoglobin.

The reactions of 13 isonitriles with deoxyhemoglobin have been examined and characterized at pH 7, 20 degrees C. Kinetic studies have shown that these ligands can be divided into two mechanistic classes based on their size and stereochemistry. The larger and branched compounds (isopropyl, all butyl isomers, n-pentyl, n-hexyl, cyclohexyl, and benzyl isocyanides) exhibit biphasic time courses at all ligand concentrations as a result of intrinsic differences between the reactivities of the alpha and beta subunits. In contrast, the smaller isonitriles (methyl, ethyl, and n-propyl isocyanides) exhibit monophasic, and often accelerating, time courses at high ligand concentrations. At low concentrations, the smaller isonitriles also exhibit biphasic time courses; however, in this case, the two phases are due to marked differences between the dissociation rate constants of the high and low affinity quaternary conformations of the protein. Sets of equilibrium and kinetic data for the binding of 11 of the isonitriles were fitted to an expanded version of the two-state allosteric model first described by Monad, Wyman, and Changeux (Monod, J., Wyman, J., and Changeux, J.-P. (1965) J. Mol. 12, 108 118). The resultant rate and equilibrium constants for the R (high affinity) and T (low affinity) states were used to calculate chemical potentials for ligand molecules bound to the heme iron and for the kinetic barriers experienced by these compounds during the binding process. For the beta subunits, both the difference between the bound chemical potentials for the R and T states and the differences between the barrier potentials for the two protein conformations are independent of ligand length and stereochemistry. Thus, it would appear that steric interactions are not a major factor in the expression of cooperativity by these subunits. For alpha chains, a 30% decrease in the difference between the bound R and T state potentials is observed in going from methyl to n-hexyl isocyanide. In addition, a marked increase in the height of the T state, alpha chain kinetic barrier is observed with increasing length of the alkyl side chain. Thus, steric hindrance between the bound ligand molecule and protein residues at the sixth coordination position of the heme iron atom does appear to play a significant role in the expression of cooperativity by the alpha subunits, at least for the larger alkyl isocyanides.