Proton NMR study of labile proton exchange in the heme cavity as a probe for the potential ligand entry channel in myoglobin

Abstract

The exchange rates of heme cavity histidine nitrogen-bound protons in horse and dog metcyanomyoglobins have been determined at 40.degree. C as a function of pH by 1H NMR spectroscopy. They were compared to the results reported for the sperm whale homologue [Cutnell, J. D., La Mar, G. N., and Kong, S. B. (1981) J. Am. Chem. Soc. 103, 3567-3572]. The rate profiles suggest that the exchange follows EX2-type kinetics, and the relative rate values favor a penetration model over a local unfolding model. It was found that the behaivor of protons located on the proximal side of the heme is similar in the three proteins. The distal histidyl imidazole NH, however, shows a highly accelerated hydroxyl ion catalyzed rate in horse and dog myoglobins relative to that in sperm whale myoglobin. NMR spectral and relaxational characteristics and the assigned heme cavity protons indicate that the global geometry of the heme pocket is highly conserved in the ground-state structure of the three proteins. We propose a model that attributes the different distal histidine exchange behavior to the relative dynamic stability of the distal heme pocket in dog or horse myoglobin vs. sperm whale myoglobin. This model involves a dynamic equilibrium between a closed heme pocket as found in metaquomyoglobin [Takano, T. (1977) J. Mol. Biol. 110, 537-568] and an open pocket as found in phenylmetmyoglobin [Ringe, D., Petsko, G. A., Kerr, D.E., and Ortiz de Montellano, P. R. (1984) Biochemistry 23, 2-4]. The greater stabilization of the open pocket in horse and dog myoglobins relative to sperm whale myoglobin is rationalized by the substitution CD3 Arg (sperm whale) .fwdarw. Lys (horse, dog). This residue holds the pocket closed in sperm whale myoglobin via hydrogen bonds to both a heme propionate and Asp E3; the substituted Lys is not capable of forming both of those stabilizing interactions in dog and horse myoglobins. Our results indicate that labile proton exchange of histidine can serve as a general useful probe of the mechanism of interaction between solvent and buried side-chain sites in folded proteins.