Masao Ikeda-Saito

Resonance Raman evidence of chloride binding to the heme iron in myeloperoxidase

The resonance Raman spectra of ferric derivatives of myeloperoxidase at pH 8 show ligand‐dependent differences. The data are consistent with the resting enzyme and the chloride and fluoride derivatives all having 6‐coordinated high‐spin configurations. At pH 4 we find that the resting enzyme is susceptible to photodegradation from our low power incident laser beam. Chloride binding inhibits this denaturation. Our data support direct binding of chloride to the enzyme under physiological conditions.

Allosteric effects in cobaltohaemoglobin as studied by precise oxygen equilibrium measurements

Accurate oxygen equilibrium curves of cobalt-substituted haemoglobin (cobalto-haemoglobin) were determined by the automatic recording method of Imai et al. (1970) in a wide range of pH values and in the presence and absence of allosteric effectors such as chloride ion, 2,3-diphosphoglycerate, inositol hexaphosphate, and orthophosphate. Cobaltohaemoglobin preserves a full Bohr effect in accordance with earlier observations. As in ferrohaemoglobin, the Adair constants ( k 1 , i =1 to 4), which measure the oxygen affinity for the four individual oxygenation steps, depend non-uniformly on pH, indicating that the number of protons released upon oxygenation differs for each step. The effectors altered the overall oxygen affinity, the co-operativity of oxygen binding, and the Adair constants for cobaltohaemoglobin. As in ferrohaemoglobin, chloride ion, 2,3-diphosphoglycerate, and orthophosphate lower the overall oxygen affinity and increase the co-operativity by reducing k 1 , k 2 and k 3 without causing significant change of k 4 , while inositol hexaphosphate lowers the overall oxygen affinity by reducing all k values. In the absence of inositol hexaphosphate, the k 4 values are similar to the oxygen association constants for free chains, implying that cobaltohaemoglobin becomes free of structural constraints prior to the last step of oxygenation. Analysis according to the allosteric model of Monod et al. (1965) indicates that the metal substitution of cobalt for iron causes a drastic shift of the allosteric equilibrium towards the R structure and an increase of the oxygen affinity of the T structure. The present results are consistent with the distributed energy model of Hopfield (1973) and, by implication, support the trigger mechanism of Perutz (1970) for co-operative oxygen binding.

Studies on cobalt myoglobins and hemoglobins X. Determination of microscopic oxygen-equilibrium constants of iron--cobalt hybrid hemoglobins and their parent hemoglobins.

Oxygen equilibrium curves of tetrameric hemoglobin, α (Fe) 2 β (Co) 2 , carrying ferrous protoporphyrin in the α subunits and cobaltous protoporphyrin in the β subunits, its complementary form α (Co) 2 β (Fe) 2 and their parent hemoglobins, i.e. Fe-hemoglobin ( α (Fe) 2 β (Fe) 2 ) and Co-hemoglobin ( α (Co) 2 β (Co) 2 ), were measured at different pH values between 6·5 and 7·9 by the automatic recording method. Each hybrid hemoglobin gave two equilibrium curves, one for Fe-containing subunits and the other for Co-containing subunits, which were measured at the isosbestic points of Co-porphyrin and Fe-porphyrin, respectively. Generalized Adair equations in which the α and β subunits were treated as non-equivalent were fitted simultaneously to the six equilibrium curves by a least-squares method to estimate the values of 12 microscopic equilibrium constants, eight of which are independent. It was assumed that interactions between the subunits depend on their ligation state but not on the kind of metal carried by them. From these parameter values the oxygen saturation of the α and β subunits were calculated as a function of average saturation and principal pathways of oxygenation on the microscopic oxygenation scheme were derived. In Fe-hemoglobin the β subunits have higher oxygen affinities than the α subunits, the difference becoming smaller as pH decreases. In Co-hemoglobin the affinity difference is slight, the α subunits having somewhat higher affinities. The present results on the affinity difference between subunits were compared with earlier conclusions drawn from other kinds of experiment. The results obtained so far are not mutually consistent and inherent problems are discussed.

Oxygen equilibrium studies on hemoglobin from the bluefin tuna (Thunnus thynnus)

Oxygen equilibrium curves of the purified hemoglobin component I from the Atlantic bluefin tuna (Thunnus thynnus) have been determined between pH 6.5 and 8.75 at 25 degrees C, and for five temperatures between 10 and 30 degrees C at pH 7.0 and 7.5. From the equilibrium data oxygen equilibrium constants for four oxygenation steps, Ki (i = 1 to 4) were estimated. The number of the Bohr protons released on the ith oxygenation (delta Hi+), and the enthalpy and entropy changes at each oxygenation step (delta Hi and delta Si) were calculated. The Hill plot for oxygenation below neutral pH is biphasic; the top asymptote lies to the right of the bottom one and the linking limb between them exhibits a slope less than unity, exhibiting apparent negative co-operativity. The values of K1 and K2 exhibit little pH dependence, while those of K3 and K4 increase by two orders of magnitudes as the pH is changed from 6.5 to 8.75. In consequence, oxygen equilibrium above neutral pH exhibits a normal positive co-operativity. The oxygen equilibrium at lower temperature is biphasic as is that below neutral pH. The shape of the Hill plot is temperature-dependent. The affinity at low saturation decreases, and that at high saturation increases upon raising the temperature from 10 to 30 degrees C, resulting in crossing of the middle portion of the equilibrium curves at different temperatures. The delta H1 and delta H2 values are negative as are those of most other hemoglobins, but the delta H3 and delta H4 values are positive. Consideration of these results in a framework of the allosteric model extended to take account of differences between subunits has indicated that the deoxy quaternary structure is stabilized at low pH or low temperature, and that subunit heterogeneity gives rise to the biphasic oxygen equilibrium curve. An analysis of delta Hi+ suggests that the large number of the Bohr groups is responsible for the biased allosteric equilibrium towards the deoxy quaternary structure. The positive delta H3 and delta H4 values are also considered to arise from the large endothermic contribution of the Bohr protons released at the third and fourth steps of oxygenation.

Subunit heterogeneity in the structure and dynamics of hemoglobin: A transient Raman study

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Studies on cobalt myoglobins and hemoglobins: XI. The interaction of carbon monoxide and oxygen with α and β subunits in iron-cobalt hybrid hemoglobins

Abstract An artificial hybrid hemoglobin, α(Co)2β(Fe)2, the α and β subunits of which carry cobaltous protoporphyrin IX and ferrous protoporphyrin IX, respectively, and its complementary hybrid α(Fe)2β(Co)2 were prepared and the properties of their ferrous subunits were examined by equilibrium and kinetic measurements with carbon monoxide as a ligand. The β(Fe)2 subunits in fully deoxy α(Co)2β(Fe)2 exhibited a higher affinity for carbon monoxide than did the α(Fe)2 subunits in fully deoxy α(Fe)2β(Co)2. Addition of 2 m m -inositol hexaphosphate decreased fourfold the carbon monoxide affinity of the α(Fe)2 subunits in α(Fe)2β(Co)2 and by more than tenfold that of the β(Fe)2 subunits in α(Co)2β(Fe)2 at pH 7.0. The higher affinity for carbon monoxide of the β(Fe)2 subunits inα(Co)2β(Fe)2 than that of the α(Fe)2 subunits in α(Fe)2β(Co)2 was caused by a smaller dissociation rate of the β(Fe)2-carbon monoxide complex. These results are considered to underlie the different affinity for carbon monoxide of the α and β subunits in deoxy hemoglobin. Oxygen equilibria of the cobaltous subunits in iron-cobalt hybrid hemoglobins were also measured in the presence of carbon monoxide. The α(Co)2 subunits in α(Co)2β(FeCO)2 showed a higher oxygen affinity than the β(Co)2 subunits in α(FeCO)2β(Co)2. Inositol hexaphosphate lowered the oxygen affinity of the β(Co)2 subunits in α(FeCO)2β(Co)2 by eight-fold, but that of the α(Co)2 subunits in α(Co)2β(FeCO)2¦by only 1.6-fold. The magnitude of the alkaline Bohr effect, as defined by Δlog P m Δ pH was found to be −0.34 and −0.14 for α(FeCO)2β(Co)2 and α(Co)2β(FeCO)2, respectively, in 0.1 m -phosphate buffer at 15 °C. The rate of oxygenation and deoxygenation of the cobaltous subunits in iron-cobalt hybrid hemoglobins in the presence of carbon monoxide was determined by a temperature-jump relaxation method in 0.1 m -phosphate buffer with and without inositol hexaphosphate. Their relaxation spectra were of a single exponential character and differed from that of cobalt hemoglobin. Without inositol hexaphosphate, the association rate constants for both α(Co)2β(FeCO)2 and α(FeCO)2β(Co)2 were close to that for cobalt hemoglobin, whereas the dissociation rate constants for iron-cobalt hybrid hemoglobins were smaller than that for cobalt hemoglobin by more than fourfold. Inositol hexaphosphate affected both the association and dissociation rates of α(FeCO)2β(Co)2 but did so to a lesser extent than those of α(Co)2β(FeCO)2. These observations suggest strongly a different role for the α and β subunits in the co-operative oxygenation and alkaline Bohr effect of hemoglobin.

Oxygenation and EPR spectral properties of Aplysia myoglobins containing cobaltous porphyrins

Cobalt myoglobins (Aplysia) have been reconstituted from apo-myoglobin (Aplysia) and proto-, meso-, and deutero-cobalt porphyrins. Each of them showed the 30--60 times lower oxygen affinity than those of the corresponding cobalt myoglobins (Sperm whale). Kinetic investigation of their oxygenation by the temperature-junp relaxation technique showed that the low oxygen affinity of cobalt myoglobin (Aplysia) is due to a large dissociation rate constant. the electron paramagnetic resonance (EPR) spectrum of oxy cobalt myoglobin (Aplysia) is affected by the replacement of H2O with D2O, suggesting a possible interaction between the bound oxygen and the neighboring hydrogen atom. A low temperature photodissociation study showed that the product of photolysis of oxy cobalt myoglobin (Aplysia) gives an EPR spectrum different from that of the deoxy-cobalt myoglobin (Aplysia) and from that of the photolysed form of oxy-cobalt myogloin (Sperm whale). These observations suggest that in oxy-cobalt myoglobin (Aplysia) the bound oxygen might interact with amino acid adjacent to it, but the interaction is weaker than that in oxy cobalt myoglobin (Sperm whale).

Characterization of the spleen green hemeprotein with magnetic and natural circular dichroism spectroscopy: positive evidence for a myeloperoxidase-type active site

The green hemeprotein purified from bovine spleen has been characterized with magnetic and natural circular dichroism (MCD and CD) spectroscopy for the first time. The enzyme derivatives studied include the native high-spin ferric form and its high-spin chloride and low-spin cyanide and nitrite complexes, the ligand-free high-spin ferrous form and its low-spin CO adduct, and Compounds II (ferryl iron species) and III (dioxygen adduct). All these enzyme states exhibit MCD spectra that are considerably different from the spectra of analogous complexes of normal heme iron. In particular, the following distinctions have been observed. The sign of the derivative-shaped MCD bands of the high-spin ferric and Compound II forms in the Soret (380-500 nm) region and of the ferrous low-spin and Compound III forms in both the Soret and visible (500-700 nm) regions are opposite to and, except for the high-spin ferric form, are less symmetric than those seen for normal heme iron systems. MCD intensities in the Soret region for the high-spin ferrous and low-spin ferric derivatives are noticeably smaller than those of normal heme proteins by a factor of up to ten. Prominent MCD bands are seen around 450 and 630 nm for the green hemeprotein derivatives; these features are considerably red-shifted (30-50 nm) relative to the analogous transitions observed for normal heme proteins. In contrast to the aforementioned spectral differences, the MCD and CD spectra of the spleen green hemeprotein derivatives are essentially identical to those previously reported for several derivatives of another spectroscopically anomalous heme-type enzyme, myeloperoxidase. This provides strong evidence that the two enzymes have identical prosthetic groups and endogenous axial ligands coordinated to the central iron. The novel MCD features of the green proteins, taken together with previously reported spectroscopic results, are most consistent with the presence of a chlorin-type prosthetic group in both proteins. In addition, the CD spectral similarities suggest that the two green proteins have nearly identical active-site environments.

[9] Preparation of hybrid hemoglobins with different prosthetic groups

Publisher Summary This chapter describes the preparation of hybrid hemoglobins with different prosthetic groups. The methods of preparation of artificial hemoglobins containing unnatural hemes or other metalloporphyrins are examined briefly. The properties of these artificial hemoglobins have to be checked before preparing the isolated chains. The oxygen-equilibrium-curve measurement is the most effective method to evaluate the quality of the preparation. An automatic-equilibrium-curve recording system—such as the Imai apparatus, which enables one to cover a wide range of saturation—is quite suitable for this purpose. As iron hemoglobins with unnatural hemes are usually prepared in the Met(Fe 3+ ) form, the metal ion has to be reduced to the ferrous state for study of their functional properties. This can be carried out easily by the use of the enzymic methemoglobin–reducing system in place of sodium dithionite or sodium borohydride. The preparation of −pMB chains are also described in the chapter.

Electron paramagnetic resonance and spectrophotometric studies of the peroxide compounds of manganese-substituted horseradish peroxidase, cytochrome-c peroxidase and manganese-porphyrin model complexes

Peroxide compounds of manganese protoporphyrin IX and its complexes with apo-horseradish peroxidase and apocytochrome-c peroxidase were characterized by electronic absorption and electron paramagnetic resonance spectroscopies. An intermediate formed upon titration of Mn(III)-horseradish peroxidase with hydrogen peroxide exhibited a new electron paramagnetic resonance absorption at g = 5.23 with a definite six-lined 55Mn hyperfine (AMn = 8.2 mT). Neither a porphyrin pi-cation radical nor any other radical in the apoprotein moiety could be observed. The reduced form of Mn-horseradish peroxidase, Mn(II)-horseradish peroxidase, reacted with a stoichiometric amount of hydrogen peroxide to form a peroxide compound whose electronic absorption spectrum was identical with that formed from Mn(III)-horseradish peroxidase. The electronic state of the peroxide compound of manganese horseradish peroxidase was thus concluded to be Mn(IV), S = 3/2. Mn(III)-cytochrome-c peroxidase reacted with stoichiometry quantities of hydrogen peroxide to form a catalytically active intermediate. The electronic absorption spectrum was very similar to that of a higher oxidation state of manganese porphyrin, Mn(V). Since the peroxide compound of manganese cytochrome-c peroxidase retained two oxidizing equivalents per mol of the enzyme (Yonetani, T. and Asakura, T. (1969) J. Biol. Chem. 244, 4580-4588), this peroxide compound might contain an Mn(V) center.