Effects of pressure on visible spectra of complexes of myoglobin, hemoglobin, cytochrome c, and horse radish peroxidase.

Abstract

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...

The effects of pressure on yeast cytochrome c peroxidase

“…The alterations in the CcP(FeII1) spectrum, seen in Fig. l A , are similar to those seen at low temperature [2], and also to the pressure-induced changes in the spectrum of horseradish peroxidase [5].…”

“…The crystal structure of CcP indicates that a water molecule is coordinated at the sixth axial position of the iron 115, 161; it also reveals a distal histidine in the heme pocket. Thus, as was previously postulated for metmyoglobin [5], the pressure-induced spectral changes may well involve substitution of the aquo ligand at the sixth coordination position by the imidazole group of the distal histidine.…”

“…In the equilibrium, the contribution of both has been determined spectrally using a pair of simultaneous equations and the following absorption coefficients : high-spin form, &408 = 95 mM-' cm-', E416. 5 7 79 mM-' cm-'; lowcm-'. The absorption coefficients were obtained from spectra of CcP(FeII1) under limiting conditions: (a) the totally highspin spectrum of CcP(FeII1) is given by the enzyme at 30"C, 1 bar, pH 6.0; (b) the totally low-spin spectrum is exhibited at -22"C, 3 kbar, pH 7.57.…”

“…1A) is characteristic of low-spin ferric heme. A I/" for the spin equilibrium is about 30 ml/mol, considerably higher that for than horseradish peroxidase [5] and cytochrome c oxidase [lo].…”

Increase in apparent compressibility of cytochrome c upon oxidation.

“…At pH 7 no technique has revealed a thermally induced transition below 53°C for either oxidation state. The visible spectra of the two forms at pH 7.0 and 25°C are unaffected by hydrostatic pressure up to 8000 kg cm-2 (780 MPa) (37 (38).…”