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 degrees 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., & 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 behavior 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 of 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., & Ortiz de Montellano, P. R. (1984) Biochemistry 23, 2-4].(ABSTRACT TRUNCATED AT 250 WORDS)

NMR investigation of ferricytochrome c unfolding: Detection of an equilibrium unfolding intermediate and residual structure in the denatured state

“…Integration was performed by using 3 mM CNmyoglobin as an external standard in a coaxial insert. Integrals of native and non-native heme methyl peaks in varying concentrations of urea-d 4 were related to the integrals of the native heme methyl peaks in the absence of urea via the resolved CN-myoglobin heme 5-CH 3 peak (28). Measurement at each urea concentration was repeated at least three times, and the results were averaged.…”

Denaturant dependence of equilibrium unfolding intermediates and denatured state structure of horse ferricytochrome c

“…The populations of non-native species detected in 3.5 M GuHCl relative to total amount of h-cyt c in the sample were determined by comparing integrals of heme methyl resonances for denatured h-cyt c in 3.5 M GuHCl to integrals of resonances of native h-cyt c in 50 mM NaP i . The resolved heme 5-CH 3 resonance of Mb-CN was used as a standard to compare populations between samples of native and denatured protein [31].…”

Hydrogen Bonding Modulates Binding of Exogenous Ligands in a Myoglobin Proximal Cavity Mutant

“…The rates of exchange (τ -1 ) were determined from the saturation factors using the expression where F is the intrinsic relaxation rate of the proton. The intrinsic lifetime was determined by measuring T 1 at pH 7, where the exchange rate τ -1 is much slower than the T 1 relaxation rate (18). T 1 was measured using a 180°-t-90°inver-sion-recovery sequence, with saturation of the water peak at all times except during acquisition.…”