Tetsutaro Iizuka

Analysis of thermal equilibrium between high-spin and low-spin states in ferrihemoglobin complexes

Abstract Paramagnetic susceptibilities and absorption spectra of ferrihemoglobin complexes (Hb(Fe 3+ ) · CN − , ·N 3 − , · OH − , · OCN − and · H 2 O) have been measured between liquid N 2 and room temperatures. It has been found that temperature-dependent transitions exist in squares of effective Bohr magneton numbers for these complexes except ferrihemoglobin cyanide, as seen in the case of ferrimyoglobin complexes, These transitions are explained on the basis of thermal equilibria between high-spin and low-spin states of Fe 3+ in hemes. In the case of acid ferrihemoglobin (pH 5.9) a strange behavior has been observed near the freezing point of solution. From the analysis of temperature dependence of the equilibrium constant in high spin ⇌ low spin states, energy and entropy differences between the two states are estimated. Thermodynamic quantities ΔH ° and ΔS ° are calculated by use of e and γ values for these complexes. Compensation phenomena between ΔH ° and ΔS ° are also described together with the results of ferric myoglobin and cytochrome c peroxidase complexes. The data of three hemoproteins are compared. This report is the fourth in our series of reports on thermal equilibrium between high-spin and low-spin states.

Studies on the heme environment of oxidized cytochrome b5

Abstract Cytochrome b5, with a molecular weight of about 1.2 · 104 was highly purified from pig liver. The purified oxidized cytochrome b5 was investigated by the following three methods: 1. (I) Absorption spectrophotometry at 23 °C and 77 °K. 2. (II) Electron Paramagnetic Resonance (EPR) spectroscopy at 20 °K. 3. (III) Kinetic measurements of the reaction with CN− in the temperature range from 22.25 to 46.75 °C. I and IIhhave demonstrated that: 3.1. 1. In the pH region from pH 5.0 to 11.0, oxidized cytochrome b5 is in a purely low-spin state between 23 °C and 20 °K. 3.2. 2. Below pH 4.0, the spin state reversibly changes to high-spin between 23 °C and 20 °K. This high-spin state is found to be due to the hemin released from cytochrome b5. 3.3. 3. Above pH 12.0, the spin state reversibly changes to another type of low-spin state between 23 °C and 20 °K, which is thought to come from a distorted protein structure but not from the liganding of OH−. 3.4. 4. Energy for three t2g orbitals calculated in one hole formalism shows a high symmetry of ligand coordination for the low-spin state at pH 6.2 and a lowering of the symmetry for another type of low-spin state at pH 12.0. III has demonstrated that 3.5. 5. The reaction with CN− is bi-phasic. The fast reaction in the cytochrome b5 monocyanide complex formation, and the slow one is the hemin dicyanide complex formation. 3.6. 6. The activation energy for fast and slow reactions are both 25.1 kcal mole. The values of entropy of activation for fast and slow reactions are 12.1 and 11.5 entropy units, respectively. The protein structure of cytochrome b5 in comparison with that of cytochrome c based on the results above as well as those of X-ray studies by Dickerson, R. E., Takano, T., Eisenberg, D., Kallai, O. B., Samoson, L., Cooper, A. and Margoliash, E. (1971) J. Biol. Chem. 246, 1511–1535 and Mathews, F. S., Levine, M. and Argos, P. (1972) J. Mol. Biol. 64, 449–464 are discussed.

NMR studies of hemoproteins. VI. Acid-base transitions of ferric myoglobin and its imidazole complex.

220 MHz proton NMR was applied to the acid-base transition of ferric myoglobin and its imidazole complex. In horse and sperm whale ferric myoglobins: (1) pH-dependent shift of heme-ring methyl signals above p2H 10 was analyzed on the basis of rapid exchange between alkaline and acidic forms by the use of pK value 9.1 of acid-base transition in 1H20 solution; (2) limiting shifts of three methyl signals were reasonably determined for purely alkaline form. For the imidazole complex: (3) a drastic high field shift of each signal was observed above p2H 9.0, whereas N0methyl imidazole complex did not exhibit such a shift, which suggests the 2H+ dissociation from liganded imidazole greater than N2H. It is concluded thns.

Kinetic study of isomerization of ferricytochrome c at alkaline pH

Kinetic studies of the isomerization reaction of horse heart ferricytochrome c between pH 8.5 and pH 12.1 have been carried out by using stopped-flow and rapid scanning stopped-flow techniques. Below pH 10, our results were in good agreement with the scheme proposed earlier (Davis, L. A., Schejter, A. and Hess, G. P. (1974) J. Biol. Chem. 249, 2624–2632). Above pH 10, another faster first-order process was observed, which suggested the existence of a transient species in the isomerization reaction between the species with and without a 695 nm band. The probable scheme of the isomerization reaction is considered to be where H denotes a proton, the colored forms are the species predominant at neutral pH with a 695 nm band and the noncolored forms are the species without a 695 nm band. The transient species has a small 695 nm absorbance which suggests that the sixth ligand is still Met-80, although the protein conformation might be different from that at neutral pH.

220 MHz proton NMR studies of hemoproteins High-spin-low-spin equilibrium in ferric myoglobin and hemoglobin derivatives

Abstract The proton NMR spectra at 220 MHz were observed at various temperatures for azide, imidazole, and deuteroxide complexes of sperm whale and horse myoglobin, and for azide and imidazole complexes of human hemoglobin. For myoglobin complexes, the size of the observed hyperfine shift and signal width increased as follows: cyanide Hyperfine shifts of these samples below −10 ppm showed abnormal temperature dependence, except purely low-spin complexes such as the imidazole complex of human hemoglobin and cyanide complexes of myoglobin and hemoglobin. These abnormal shifts were discussed and analyzed on the basis of the spin equilibrium of the heme iron confirmed previously by magnetic susceptibility measurements.

Studies on the charge transfer band in high spin state of ferric myoglobin and hemoglobin by low temperature optical and magnetic circular dichroism spectroscopy.

The behavior of charge transfer band, appearing at 600-650 nm in ferric high spin derivatives of myoglobin and hemoglobin, was studied under various conditions by low temperature optical and magnetic circular dichroism spectroscopy. Optical absorption spectra have demonstrated that: (1) The charge transfer band at 630 nm of myoglobin (Fe3+)-H2O (pH 7.0) at room temperature split into three bands, 627 nm, 645 nm and 664 nm (shoulder) at 77 degrees K, whereas that of hemoglobin (Fe3+)-H2O showed no splitting. (2) By lowering the pH value from 7.5 to 4.3 this splitting in myoglobin was observed to disappear only in the presence of a small amount of phosphate ion, accompanying a midpoint at pH 6.7 +/- 0.1. This does not originate from the released hemin. (3) Hemin (pH 7.55) showed no splitting of the charge transfer band at 77 degrees K. (4) This splitting depended on the species of 6th ligand. For myoglobin-F- the splitting could scarcely be observed, whereas the proton-donating ligands such as HCOOH and CH3OH exhibit the splitting as well as H2O. Magnetic circular dichroism spectra have demonstrated that: (5) The charge transfer band at 600-500 nm indicated Faraday A term and B term. (6) A negative B term band was observed at 650 nm for myoglobin-H2O in the glassic solvent of potassium glycerophosphate-glycerol, whereas it was not observed for hemoglobin-H2O. Several discussions were performed on the origin of splitting of the charge transfer band in myoglobin-H2O. It is now concluded that the hydrogen bond between the 6th ligand and the distal histidine contributes to the splitting of the charge transfer band around 630 nm for myoglobin Fe3+)-H2O at low temperature and that disappearance of the splitting at low pH is originated from the presence of phosphate ion.

NMR studies of hemoproteins: pH dependence of ferric horseradish peroxidase and horse heart myoglobin

N.m.r. spectroscopy has recently developed into a powerful tool for investigating structure and structurefunction relationships of hemo-proteins and heme enzymes. Most of the n.m.r. studies on ferric hemoproteins so far reported have been performed with those in purely or nearly low spin state. Several of the ferric hemoproteins, which exhibit a pHdependent spin state change, have been incompletely investigated [ 1,2] . In this report we concentrate on the n.m.r. measurements of pH-dependence for ferric horseradish peroxidase and horse heart myoglobin, covering the large hyperfine shift of heme ring methyl groups due to ferric high spin state. 220 MHz proton n.m.r. confirmed the result of magnetic susceptibility measurements by Theorell and Ehrenberg [3], and was, furthermore, able to distinguish the nature of the pH dependent spin state change between both hemoproteins.

Studies on the heme environment of horse heart ferric cytochrome c. Azide and imidazole complexes of ferric cytochrome c.

Horse heart ferric cytochrome c was investigated by the following three methods: (I) Light absorption spectrophotometry at 23 degrees C and 77 degrees K; (II) Electron paramagnetic resonance (EPR) spectroscopy at 20 degrees K; (III) Precise equilibrium measurements of ferric cytochrome c with azide and imidazole between 14.43 and 30.90 degrees C. I and II have demonstrated that: (1) Ferric cytochrome c azide and imidazole complexes were in the purely low spin state between 20 degrees K and 23 degrees C; (2) The energy for the three t2g orbitals calculated in one hole formalism shows that azide or imidazole bind to the heme iron in a similar manner to met-hemoglobin azide or imidazole complexes, respectively. III has demonstrated that: (1) The change of standard enthalpy and that of standard entropy were -2.3 kcal/mol and -1.6 cal/mol per degree for the azide complex formation, and -1.4 kcal/mol and 2.9 cal/mol per degree for the imidazole complex formation. (2) A linear relationship between the change of entropy and that of enthalpy was observed for the above data for the cyanide complex formation. The complex formation of ferric cytochrome c was discussed based on the results of X-ray crystallographic studies compared with hemoglobin and myoglobin.

Resonance Raman scattering from hemoproteins: pH-dependence of Raman spectra of ferrous dicarboxymethyl—methionyl—cytochrome C

The pH-dependent change of absorption spectra of ferrous alkylated cytochrome c (dicarboxymethylmethionyl-cytochrome c) was reported to be due to the transition between low spin and high spin states [ 11. Since the ferrous cytochrome c gives strong Raman lines [2] and Raman spectra are sensitive to the spin and oxidation states [3]. of hemoproteins, we attempted to measure the resonance Raman spectra as well as optical absorption spectra of ferrous alkylated cytochrome c in the region between pH 3.0 and 11 .O. General patterns of Raman spectra of ferrous alkylated cytochrome c resemble to that of ferrous native cytochrome c and most of Raman lines do not + show pH-dependent frequency shift. However, a pHsensitive Raman line was found around 1540 cm’ and it was concluded that the pH-dependent change is due to the transition between two kinds of low spin state with a pK of 7.9. In alkylated cytochrome c two methionyl residues (at the 65th and 80th positions) were alkylated with bromoacetate in the presence of KCN [4], and lysine-79 is supposed to be the 6th ligand of the heme iron [ 51. Since ferrous native cytochrome c did not show any pH-dependence of Raman spectra, the spectral change of alkylated cytochrome c is deduced to be associated with the coordination of lysine-79. The spin state and structure of ferrous alkylated cytochrome c will be discussed

Nuclear magnetic resonance studies of hemoproteins IV. Hindered rotation of heme side methyl group as a probe for studying van der waals contacts in the heme side environments of myoglobin derivatives

220 MHz roton NMR spectral evidence for restricted rotation of one methyl group in the heme side chain of ferric horse cyanomyoglobin is reported here. Temperature dependence of this methyl proton signal was computer-simulated, yielding 14,8 kcal/mol for the methyl hindered rotation. Ionic additives such as NaCl and (NH4) 2 minus SO4 caused a slackening of this restriction of methyl rotation, evidenced from collapse of methyl signal doubling by the addition of these ionic substances. This is discussed in terms of breaking of the salt bridge formed between one of the propionate COO minus group of heme and a part of the apoprotein which might lead to constraint of one of the heme side methyl groups. The peculiarity of hyperfine-shifted methyl proton signals for other myoglobin complexes such as azide and imidazole derivatives is also discussed briefly in terms of constraint of heme side methyl group buried in a hydrophobic cleft.