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

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Abstract
Hyperfine shifted proton NMR spectra of [sperm whale] metmyoglobin (metMb), [human] met-Hb, 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 metMb and met-Hb and the imidazole complex of metMb, in all of which the Fe 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 metMb and met-Hb and for the fluoride complex of metMb, 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 aquometMb, its formate complex, and horseradish peroxidase showed appreciable upfield shifts upon pressurization. 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 met-Hb at high pressures was also observed, and the effect of pressure on the heme environmental structure of deoxy-Hb and deoxyMb was also discussed.