Proton nuclear magnetic resonance study of the dynamic stability of the heme pocket of soybean leghemoglobin a. Exchange rates for the labile proton of the proximal histidyl imidazole.

Intrinsic spin lattice relaxation times for the hyperfine-shifted exchangeable resonances and the downfield heme methyls for the low spin met-cyano-, met-nicotinate-, and deoxy- complexes of soybean leghemoglobin a were determined in H2O. When exchange with the bulk solvent is slow on the T1 time scale, comparison of the intrinsic T1 values for the exchangeable protons and the heme methyl resonances has provided the assignment of the proximal histidyl imidazole N1H proton resonance. Transfer of saturation experiments and linewidth data as a function of pH at 25 degrees C permitted the determination of the proximal histidyl imidazole N1H exchange rates in various protein oxidation/ligation states. The exchange rates were found to be base-catalyzed in the deoxy- as well as the met-cyano-, met-nicotinate-, and met-azide- complexes. The exchange rates are taken as measures of the magnitude of the fluctuations of the protein conformation near the heme cavity. The unligated deoxy-protein exhibited a greater kinetic stability than ligated forms of the protein, and the bulky nicotinate ligand resulted in the lowest kinetic stability for the ligated protein forms. In contrast to met-cyanomyoglobin, no resonance which could be attributed to the distal histidyl imidazole NH was observed for any of the low spin complexes of leghemoglobin. Comparison between the same form of leghemoglobin and myoglobin reveals that the former exhibits exchange rates an order of magnitude faster than the latter protein in both ligated and unligated states, confirming the greater flexibility of the heme pocket in leghemoglobin.