High-pressure proton nuclear magnetic resonance studies of hemoproteins. Pressure-induced structural change in heme environments of myoglobin, hemoglobin, and horseradish peroxidase.

Hyperfine shifted proton NMR spectra of metmyoglobin, methemoglobin, and their complexes with azide, imidazole, and cyanide as well as the spectrum of native horseradish peroxidase were obtained at high pressures up to 2000 atm with a specially designed high-pressure cell for 220-MHz superconducting NMR spectrometer. For the azide complexes of metmyoglobin, in all of which the iron atoms are in thermal spin equilibrium between high- and low-spin states, the increased pressure shifted their heme methyl proton signals to the upfield side. For the cyanide complexes of metmyoglobin and methemoglobin and for the fluoride complex of metmyoglobin, which are in purely low- and high-spin states, respectively, the spectra were almost insensitive to changes in pressure up to 2000 atm. The heme methyl proton signals of aquometmyoglobin, its formate complex, and horseradish peroxidase showed appreciable upfield shifts upon pressurization. These results were interpreted to indicate that the primary effect of pressure on the hemoprotein structure is to shift the spin equilibrium in favor of the low-spin form. Hemichrome formation of methemoglobin at high pressures was also observed, and the effect of pressure on the heme environmental structure of deoxyhemoglobin and deoxymyoglobin was also discussed.