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.

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.

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.

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).

Interaction of halides with the cyanide complex of myeloperoxidase: a model for substrate binding to compound I.

EPR spectra of the low-spin cyanide complex of myeloperoxidase have been measured in the absence and presence of halide substrates; chloride, bromide and iodide. Halide-dependent spectral changes are found at acidic pH. The electronic structure of the low-spin ferric iron in cyanide complex appears to be modulated by halide binding to a protonated amino acid in the distal heme cavity. These findings suggest halide substrates can interact with ferryl oxygen in compound I during enzyme catalysis to form hypohalous acid.

Cobalt-substituted hemoglobin Zürich (alpha 2 beta 263His leads Arg). Oxygen equilibria and EPR spectra.

Cobalt hemoglobin Zürich (alpha 2 beta 263His leads to Arg) has been successfully reconstituted from the apohemoglobin Zürich and cobaltous protoporphyrin IX. The oxygen affinity of cobalt hemoglobin Zurich, as well as that of iron hemoglobin Zürich, were measured in the absence and presence of organic phosphate and Cl-. The overall oxygen affinity of cobalt hemoglobin Zürich was found to be higher and the cooperativity as measured by the n value was smaller than those of cobalt hemoglobin A. Organic phosphate and Cl- affect the oxygen equilibrium properties of cobalt hemoglobin Zürich in a manner similar to that of cobalt hemoglobin A, but to a lesser extant than cobalt hemoglobin A. The EPR spectrum of oxy cobalt hemoglobin Zürich is less sensitive to the replacement of the buffer system from H2O to 2H2O, indicating that the hydrogen bond between the distal amino acid residue and the bound oxygen is not formed in the abnormal beta subunits. The deoxy EPR spectrum of cobalt hemoglobin Zürich is similar to that of deoxy cobalt hemoglobin A, suggesting that the deoxy cobalt hemoglobin Zürich is predominantly in the deoxy quaternary structure (T state).

Studies on Scapharca hemoglobins. Properties of the dimeric protein reconstituted with Fe- or Co-porphyrin.

A native globin from the dimeric hemoglobin, hemoglobin I, of the mollusc Scapharca inaequivalvis has been obtained with the acid-acetone method. The globin has a lower sedimentation coefficient than the native protein at neutral pH; its reconstitution product with natural heme has the same physicochemical and functional properties as the native protein. proto- and meso-cobalt hemoglobin I have been prepared and characterized. proto-Cobalt hemoglobin I binds oxygen reversibly with a lower affinity and a lower cooperativity than native hemoglobin I; thus, the changes in the functional properties brought about by substitution of iron with cobalt are similar to those observed in human hemoglobin A. The EPR spectra of deoxy-proto-cobalt hemoglobin I and of the photolysis product of oxy-meso-cobalt hemoglobin I indicate that two histidine residues are the apical heme ligands. The broad signal at g = 2.38 in deoxy-proto-cobalt hemoglobin I points to a constrained structure of the heme site in this derivative which results from a distorted coordination of the hindered proximal histidine. A similar structure has been proposed previously for the alpha chains in deoxy-cobalt hemoglobin A.

African elephant myoglobin with an unusual autoxidation behavior: comparison with the H64Q mutant of sperm whale myoglobin.

Elephant myoglobins both from Asian and African species have a glutamine in place of the usual distal (E7) histidine at position 64. We have isolated native oxymyoglobin directly from the skeletal muscle of African elephant (Loxodonta africana), and examined the autoxidation rate of oxymyoglobin (MbO2) to metmyoglobin (metMb) as a function of pH in 0.1 M buffer at 25 degreesC. As a result, African elephant MbO2 was found to be equally resistant to autoxidation as sperm whale myoglobin. However, the elephant myoglobin exhibited a distinct rate saturation below pH 6. Kinetic analysis of the pH profiles for the autoxidation rate has disclosed that African elephant MbO2 does not show any proton-catalyzed process, such as the one that can play a dominant role in the autoxidation reaction of sperm whale myoglobin by involving the distal histidine as its catalytic residue. Such a greater stability of African elephant MbO2 at low pH could be explained almost completely by the single H64Q mutation of sperm whale myoglobin. In African elephant aqua-metmyoglobin the Soret band was considerably broadened so as to produce another peak in the pentacoordinate 395 nm region. This unique spectral feature was therefore analyzed to show that the myoglobin is in equilibrium between two species, depending upon the presence or absence of a water molecule at the sixth coordinate position.

Transient kinetics of oxygen dissociation from ferrous subunits of iron-cobalt hybrid hemoglobins. The principal reaction controlling the co-operativity.

The oxygen dissociation constants from Fe subunits in the half-ligated intermediate states of Fe-Co hybrid hemoglobins, alpha(Fe-O2)2 beta(Co)2 and alpha(Co)2 beta(Fe-O2)2, have been determined as functions of pH, temperature and inositol hexaphosphate. The oxygen dissociation rates from alpha(Fe-O2)2 beta(Co)2 are estimated to be more than 1300 s-1 for the deoxy quaternary state (T-state) and less than 3 s-1 for the oxy quaternary state (R-state) at 15 degrees C in 50 mM-Tris or Bis-Tris buffer containing 0.1 M-Cl-, while those of alpha(Co)2 beta(Fe-O2)2 are more than 180 s-1 and less than 5 s-1 for the T and R-states, respectively. The pH dependence of the oxygen dissociation rate from Fe subunits is large enough to be accounted for by the R-T transition, and implies that those half-ligated intermediate hybrids mainly exist in the R-state at pH 8.8, and in the T-state at pH 6.6, while other studies indicated that the half-ligated hybrids are essentially in the R-state at pH 7. Large activation energies of the oxygen dissociation process of 19 to 31 kcal/mol determined from the temperature dependence suggest that the process is entropy-driven.

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.

Thermodynamic properties of oxygen equilibria of dimeric and tetrameric hemoglobins from Scapharca inaequivalvis.

Oxygen equilibrium curves of the dimeric and tetrameric hemoglobin components of Scapharca inaequivalvis were determined at several temperatures. The oxygen equilibrium curves were analyzed by the two-step and four-step oxygen equilibrium schemes of Adair for the dimeric and tetrameric hemoglobins, respectively. The enthalpy and entropy changes for each oxygenation step were determined by the temperature dependences of the Adair constants using van't Hoff equations. Neither dimeric nor tetrameric hemoglobins release protons or anions upon oxygenation under the experimental conditions of the present study. The enthalpy and entropy changes are non-uniform with respect to the oxygenation step and do not need to be corrected for the oxygenation-linked release of protons and anions. For the tetrameric hemoglobin, enthalpy-entropy compensation was observed between the first, second and third steps of oxygenation. The present results suggest that the origin of the co-operative oxygenation is primarily entropic for both hemoglobin components of S. inaequivalvis. Comparison of these data with those obtained on other hemoglobins shows that no simple generalization can be made as to the thermodynamic nature of co-operativity in oxygen binding.

Structural and functional effects of apolar mutations of the distal valine in myoglobin.

High-resolution structures of the aquomet, deoxy, and CO forms of Ala68, Ile68, Leu68, and Phe68 sperm whale myoglobins have been determined by X-ray crystallography. These 12 new structures, plus those of wild-type myoglobin, have been used to interpret the effects of mutations at position 68 and the effects of cobalt substitution on the kinetics of O2, CO, and NO binding. Molecular dynamics simulations based on crystal structures have provided information about the time-dependent behavior of photolyzed ligands for comparison with picosecond geminate recombination studies. The Val68-->Ala mutation has little effect on the structure and function of myoglobin. In Ala68 deoxymyoglobin, as in the wild-type protein, a water molecule hydrogen-bonded to the N epsilon atom of the distal histidine restricts ligand binding and appears to be more important in regulating the function of myoglobin than direct steric interactions between the ligand and the C gamma atoms of the native valine side-chain. This distal pocket water molecule is displaced by the larger side-chains at position 68 in the crystal structures of Leu68 and Ile68 deoxymyoglobins. The Leu68 side-chain can rotate about its C alpha-C beta and C beta-C gamma bonds to better accommodate bound ligands, resulting in net increases in overall association rate constants and affinities due to the absence of the distal pocket water molecule. However, the flexibility of Leu68 makes simulation of picosecond NO recombination difficult since multiple starting conformations are possible. In the case of Ile68, rotation of the substituted side-chain is restricted due to branching at the beta carbon, and as a result, the delta methyl group is located close to the iron atom in both the deoxy and liganded structures. The favorable effect of displacing the distal pocket water molecule is offset by direct steric hindrance between the bound ligand and the terminal carbon atom of the isoleucine side-chain, resulting in net decreases in affinity for all three ligands and inhibition of geminate recombination which is reproduced in the molecular dynamics simulations. In Phe68 myoglobin, the benzyl side-chain is pointed away from the ligand binding site, occupying a region in the back of the distal pocket. As in wild-type and Ala68 myoglobins, a well-defined water molecule is found hydrogen bonded to the distal histidine in Phe68 deoxymyoglobin. This water molecule, in combination with the large size of the benzyl side-chain, markedly reduces the speed and extent of ligand movement into the distal pocket. (ABSTRACT TRUNCATED AT 400 WORDS)

Probing protein-cofactor interactions in the terminal oxidases by second derivative spectroscopy: study of bacterial enzymes with cofactor substitutions and heme A model compounds.

Second derivative absorption spectra are reported for the aa3-cytochrome c oxidase from bovine cardiac mitochondria, the aa3-600 ubiquinol oxidase from Bacillus subtilis, the ba3-cytochrome c oxidase from Thermus thermophilis, and the aco-cytochrome c oxidase from Bacillus YN-2000. Together these enzymes provide a range of cofactor combinations that allow us to unequivocally identify the origin of the 450-nm absorption band of the terminal oxidases as the 6-coordinate low-spin heme, cytochrome a. The spectrum of the aco-cytochrome c oxidase further establishes that the split Soret band of cytochrome a, with features at 443 and 450 nm, is common to all forms of the enzyme containing ferrocytochrome a and does not depend on ligand occupancy at the other heme cofactor as previously suggested. To test the universality of this Soret band splitting for 6-coordinate low-spin heme A systems, we have reconstituted purified heme A with the apo forms of the heme binding proteins, hemopexin, histidine-proline-rich glycoprotein and the H64V/V68H double mutant of human myoglobin. All 3 proteins bound the heme A as a (bis)histidine complex, as judged by optical and resonance Raman spectroscopy. In the ferroheme A forms, none of these proteins displayed evidence of Soret band splitting. Heme A-(bis)imidazole in aqueous detergent solution likewise failed to display Soret band splitting. When the cyanide-inhibited mixed-valence form of the bovine enzyme was partially denatured by chemical or thermal means, the split Soret transition of cytochrome a collapsed into a single band at 443 nm.(ABSTRACT TRUNCATED AT 250 WORDS)

On the analogy in the structure of the spleen green heme protein and granulocyte myeloperoxidase.

The molecular structure of the spleen green heme protein was reinvestigated by gel-permeation, SDS-polyacrylamide gel electrophoresis, and amino acid analysis. The results showed that the enzyme is a tetramer (Mr 1.5 X 10(5)) with two heavy subunits (Mr 6 X 10(4) with a single prosthetic group per subunit) and two light subunits (Mr 1.5 X 10(4)), and that the tetramer structure is maintained by disulfide bond(s). The amino acid composition of the spleen green heme protein is similar to that of granulocyte myeloperoxidase. The present results contradict the data of Davis and Averill [(1981) J. Biol. Chem. 256, 5992-5996], who reported the enzyme as a monomeric peroxidase with an Mr of 57 000.

Proton magnetic resonance of the bovine spleen green heme-protein.

The ferric spleen green heme-protein exhibits hyperfine-shifted proton resonances between 90 and 20 ppm for the high-spin resting form and the chloride complex, and between 46 and -9.4 ppm for the low-spin nitrite complex. The proton NMR spectral profile of the enzyme is similar to that of lactoperoxidase, but different from those of common heme-proteins. The appearance of a resonance at 76 ppm in the ferrous enzyme shows the presence of a proximal histidine residue linked to the iron. The proton relaxation rates of bulk water indicate that chloride binds to the sixth position of the iron in the chloride complex of the enzyme.

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.

Oxygen equilibrium and electron paramagnetic resonance studies on copper(II)-iron(II) hybrid hemoglobins at room temperature.

Copper(II)-iron(II) hybrid hemoglobins, in which hemes in either the alpha or beta subunits are substituted with copper(II) protoporphyrin IX, have been prepared. The affinities of the ferrous-subunits in both hybrids for the first binding oxygen are as low as the affinity of deoxyhemoglobin under various solution conditions, indicating the equality of behavior in copper(II) protoporphyrin IX and deoxyheme. Electron paramagnetic resonance (EPR) examinations on these hybrids at room temperature show that the interaction between copper(II) and the proximal histidine (F8) is specifically weakened in the alpha subunits within a low affinity conformation of hemoglobin. These results suggest that copper(II) protoporphyrin IX is a useful EPR probe at room temperature for investigating the deoxyheme environment in hemoglobin.

Resonance Raman scattering from hemoproteins. Effects of ligands upon the Raman spectra of various C-type cytochromes.

Resonance Raman spectra were measured for various C-type cytochromes (mammalian cytochrome c, bacterial cytochrome c3, algal photosynthetic cytochrome f, and alkylated cytochrome c) and a B-type cytochrome (cytochrome b5) in their reduced and oxidized states. (1) For ferrous alkylated cytochrome c, a Raman line sensitive to the replacement of an axial ligand of the heme iron uas found around 1540 cm=1. This ligand-sensitive Raman line indicated the transition from acidic (1545 cm-1) to alkaline (1533 cm-1) forms with pK 7.9. The pH dependence of the Raman spectrum corresponded well to that of the optical absorption spectra. (2) For ferrous cytochrome f, the ligand-sensitive Raman line was found at the same frequency as cytochrome c (1545 cm-1). Accordingly two axial ligands are likely to be histidine and methionine as in cytochrome c. (3) For ferrous cytochrome c3, the frequency of the ligand-sensitive Raman line was between those of cytochrome c and cytochrome b5. Since two axial ligands of the heme iron in cytochrome c3 might be histidines. However, a combination of histidine and methionine as a possible set of two axial ligands was not completely excluded for one or two of the four hemes. (4) In ferrous cytochrome b5, two weak Raman lines appeared at 1302 and 1338 cm-1 instead of the strongest band at 1313 cm-1 of C-type ferrous cytochromes. This suggests the practical use of these bands for the identification of types of cytochromes. The difference in frequency and intensity between B- and C-types of hemes implies that the low effective symmetry of the heme in ferrous cytochrome c is due to vibrational coupling of ring modes with peripheral substituents rather than geometrical disortion of heme.

EPR studies on the photoinduced intermediates of NO complexes in recombinant ferric-Mb trapped at low temperatures.

The nitrosyl complex of ferric myoglobin is EPR-silent. Upon photolysis at low temperatures, the photoinduced intermediates trapped in the distal heme cavity exhibit new EPR spectra due to the interaction between the photodissociated NO (S=1/2) and the ferric high spin heme (S=5/2). In order to elucidate the effect of distal E7 (His64) and E11 (Val68) mutations upon the electronic structure of the metal center, its immediate environment, and its interaction with the photodissociated NO, EPR spectra of the photoproducts of the NO complexes of recombinant ferric Mb mutants were measured at 5 K. EPR spectra of the photoproducts were closely related to the size and/or the polarity of the distal pocket residues. The distal pocket of the E7 mutants seemed to be sterically crowded, even decreasing the side chain volume or changing its hydrophobicity by replacing amino acid at position 64. We have found that the mobility of the photodissociated NO molecule in the distal heme pocket was strongly governed by the nature of the amino acid residue at E11 position.