Proton magnetic resonance characterization of the dynamic stability of the heme pocket in myoglobin by the exchange behavior of the labile proton of the proximal histidyl imidazole.

The assigned exchangeable proton signals in the proton nuclear magnetic resonance spectra of sperm whale deoxy and Met-cyano myoglobin in H2O solution were found to exhibit pH-dependent saturation transfer from the bulk water, which allowed determination of the kinetics and mechanism of the labile proton exchange with solvent. The exchange rates are base catalyzed for both protein forms, with the rate eight times faster in Met-cyano than in deoxy myoglobin. The exchange rate is taken as a measure of the magnitude of the fluctuation in the protein conformation near the heme cavity. On the basis of tritium exchange methods, the greater stability of the unligated relative to the ligated state in myoglobin has also been reported for hemoglobin. The present study, however, localizes the differential kinetic stability on the F helix whose flexibility has been implicated in the mechanism of cooperativity. The observation that filling the hydrophobic vacancy on the proximal side of the heme near the proximal histidine in Met-cyano myoglobin wih cyclopropane increases the proton lability argues against the role for this hole in facilitating the flexibility of the F helix in the native protein.