The spectra of the ferric form of most heme proteins [metmyoglobin, methemoglobin, horse radish peroxidase (EC 1.11.1.7), and ferricytochrome c at pH 1.5J are converted from high-spin (open crevice) structure to low-spin (closed crevice) form under pressure. Pressures up to 8000 kg/cm2 (780 MPa) have no effect on the spectra of high-spin ferro-and ferricytochrome c, which have a closed crevice structure at pH 7.0. Spectra of deoxy-ferromyoglobin and deoxy-ferrohemoglobin are reduced in intensity, but pressure does not change the positions of the absorption maxima. Cyanide ion prevents pressure-induced spectral changes in metmyoglobin and methemoglobin up to 8000 kg/cm2. Carbon monoxide (with a high affinity for the ferro heme iron) has a similar effect on ferromyoglobin and ferrohemoglobin. The pressure required to cause spectral changes in the heme proteins falls in the order, cytochrome c (pH 7.0) > horse radish peroxidase > myoglobin > hemoglobin. We have calculated a volume change of -50 cm:3/mol associated with the configurational change accompanying the reformation of the iron-methionine bond in cytochrome c at low pH.The effect of pressure on the complexes of hemoglobin (1-4) and myoglobin (5-7) have been the subject of considerable investigation over the last few years. These investigations have, however, been largely concerned with changes in the functional properties (1, 2), the thermodynamic properties accompanying the binding of ligands (6), and the denaturation of the protein (5). However, the Soret and visible spectra of hemoglobin and hemoglobin complexes (3, 4) and the visible spectra of metmyoglobin fluoride (7) show interesting changes when these molecules are subjected to hydrostatic pressure. We have extended these high pressure studies on metmyoglobin and methemoglobin and have investigated, in addition, the spectral changes in the visible region accompanying the pressurization of two other hemoproteins, cytochrome c and horse radish peroxidase (donor:hydrogen-peroxide oxidoreductase; EC 1.11.1.7).
MATERIALS AND METHODSHuman hemoglobin was prepared by the usual method (8) and oxidized to methemoglobin with 2-fold excess of potassium ferricyanide. Ferrocyanide and excess ferricyanide were removed by dialysis against 0.05 M sodium chloride at pH 6.0. Sperm whale metmyoglobin and horse heart (type III) cytochrome c were obtained from Sigma Chemical Co. and were used without further purification. Buffers used were cacodylate and Tris. Tris buffer was prepared from Trizma base (Sigma) and Trizma-HCI (Sigma) that had been dried under reduced pressure before use. All methemoglobin and metmyoglobin solutions were 0.05 M in buffer ions. Hemoglobin and myoglobin were deoxygenated by addition of sodium dithionite in a glove box previously filled with dry nitrogen.High Pressure Studies. The high pressure optical bomb used was designed by Dr. W. B. Daniels and has been described elsewhere (9).At each increment of pressure, the system was allowed 5 min to attain temperature re-equilibration. Me...