Aplysia oxymyoglobin with an unusual stability property: involvement of two kinds of carboxyl groups

Unlike mammalian oxymyoglobins, Aplysia MbO2 is extremely susceptible to autoxidation, and its pH dependence is also unusual. Kinetic formulation has revealed that two kinds of dissociable group with pK1 = 4.3 and pK2 = 6.1, respectively, at 25 degrees C are involved in the stability property of Aplysia MbO2. In order to characterize thermodynamically these dissociation processes involved, the effect of temperature on K1 and K2 was studied by analyzing the pH dependence for the autoxidation rate of Aplysia MbO2 in 0.1 M buffer over the pH range of 4-11, and at 15, 25 and 35 degrees C. The resulting thermodynamic parameters for each group were both those to be expected for the ionization of a carboxyl group; the delta H degrees value being numerically much less than 1 kcal.mol-1, or zero in practice, but being associated with a large negative value of delta S degrees of the order of -20 cal.mol-1.K-1. Taking into account the fact that Aplysia myoglobin contains only a single histidine residue corresponding to the heme-binding proximal one, we can unequivocally conclude that the two kinds of the dissociable group involved in the unusual stability of Aplysia MbO2 must both be carboxyl groups, the protonation of these groups being responsible for an increase in its autoxidation rate in the acidic pH range.     

Role of globin moiety in the autoxidation reaction of oxymyoglobin: effect of 8 M urea.

It is in the ferrous form that myoglobin or hemoglobin can bind molecular oxygen reversibly and carry out its function. To understand the possible role of the globin moiety in stabilizing the FeO2 bond in these proteins, we examined the autoxidation rate of bovine heart oxymyoglobin (MbO2) to its ferric met-form (metMb) in the presence of 8 M urea at 25 degrees C and found that the rate was markedly enhanced above the normal autoxidation in buffer alone over the whole range of pH 5-13. Taking into account the concomitant process of unfolding of the protein in 8 M urea, we then formulated a kinetic procedure to estimate the autoxidation rate of the unfolded form of MbO2 that might appear transiently in the possible pathway of denaturation. As a result, the fully denatured MbO2 was disclosed to be extremely susceptible to autoxidation with an almost constant rate over a wide range of pH 5-11. At pH 8.5, for instance, its rate was nearly 1000 times higher than the corresponding value of native MbO2. These findings lead us to conclude that the unfolding of the globin moiety allows much easier attack of the solvent water molecule or hydroxyl ion on the FeO2 center and causes a very rapid formation of the ferric met-species by the nucleophilic displacement mechanism. In the molecular evolution from simple ferrous complexes to myoglobin and hemoglobin molecules, therefore, the protein matrix can be depicted as a breakwater of the FeO2 bonding against protic, aqueous solvents.     

Amino acid sequence of myoglobin from the mollusc Dolabella auricularia

The complete amino acid sequence of the myoglobin from Dolabella auricularia, a common gastropodic mollusc on the Japanese coast, has been determined. The myoglobin is composed of 146 amino acid residues, is acetylated at the NH2 terminus, and contains a single histidine residue at position 95 which most likely corresponds to the heme-binding proximal histidine. The sequence of Dolabella myoglobin shows strong homology (72-77%) with those of Aplysia myoglobins. The autoxidation rate of Dolabella oxymyoglobin (MbO2) was examined in 0.1 M buffer at 25 degrees C over pH range 4.8-12. Dolabella MbO2 was extremely unstable between pH 7 and 11, and the pH dependence of the stability was quite different from that of sperm whale MbO2. This property may be partly due to the absence of a distal (E7) histidine in Dolabella myoglobin.     

Autoxidation of myoglobin from bigeye tuna fish (Thunnus obesus)

Native oxymyoglobin (MbO2) was isolated directly from the skeletal muscle of bigeye tuna (Thunnus obesus) with complete separation from metmyoglobin (metMb) on a CM-cellulose column. It was examined for its stability properties over a wide range of pH values (pH 5-12) in 0.1 M buffer at 25 degrees C. When compared with sperm whale MbO2 as a reference, the tuna MbO2 was found to be much more susceptible to autoxidation. Kinetic analysis has revealed that the rate constant for a nucleophilic displacement of O2- from MbO2 by an entering water molecule is 10-times higher than the corresponding value for sperm whale MbO2. The magnitude of the circular dichroism of bigeye tuna myoglobin at 222 nm was comparable to that of sperm whale myoglobin, but its hydropathy profile revealed the region corresponding to the distal side of the heme iron to be apparently less hydrophobic. The kinetic simulation also demonstrated that accessibility of the solvent water molecule to the heme pocket is clearly a key factor in the stability properties of the bound dioxygen.     

Dual nature of the distal histidine residue in the autoxidation reaction of myoglobin and hemoglobin comparison of the H64 mutants.

The oxygenated form of myoglobin or hemoglobin is oxidized easily to the ferric met-form with generation of the superoxide anion. To make clear the possible role(s) of the distal histidine (H64) residue in the reaction, we have carried out detailed pH-dependence studies of the autoxidation rate, using some typical H64 mutants of sperm whale myoglobin, over the wide range of pH 5-12 in 0.1 M buffer at 25 degrees C. Each mutation caused a dramatic increase in the autoxidation rate with the trend H64V >/= H64G >/= H64L > H64Q > H64 (wild-type) at pH 7.0, whereas each mutant protein showed a characteristic pH-profile which is essentially different from that of the wild-type or native sperm whale MbO2. In particular, all the mutants have lost the acid-catalyzed process that can play a dominant role in the autoxidation reaction of most mammalian myoglobins or hemoglobins. Kinetic analyses of various types of pH-profiles lead us to conclude that the distal histidine residue can play a dual role in the nucleophilic displacement of O2- from MbO2 or HbO2 in protic, aqueous solution. One is in a proton-relay mechanism via its imidazole ring, and the other is in the maximum protection of the FeO2 center against a water molecule or an hydroxyl ion that can enter the heme pocket from the surrounding solvent.     

NMR study of the alkaline isomerization of ferricytochrome c

The pH-induced isomerization of horse heart cytochrome c has been studied by 1H NMR. We find that the transition occurring in D2O with a pKa measured as 9.5 +/- 0.1 is from the native species to a mixture of two basic forms which have very similar NMR spectra. The heme methyl peaks of these two forms have been assigned by 2D exchange NMR. The forward rate constant (native to alkaline cytochrome c) has a value of 4.0 +/- 0.6 s-1 at 27 degrees C and is independent of pH; the reverse rate constant is pH-dependent. The activation parameters are delta H not equal to = 12.8 +/- 0.8 kcal.mol1, delta S not equal to = -12.9 +/- 2.0 e.u. for the forward reaction and delta H not equal to = 6.0 +/- 0.3 kcal.mol-1, delta S not equal to = -35.1 +/- 1.3 e.u. for the reverse reaction (pH* = 9.28). delta H degree and delta S degree for the isomerization are 6.7 +/- 0.6 kcal.mol-1 and 21.9 +/- 1.0 e.u., respectively.     

Autoxidation of oxymyoglobin. An overall stoichiometry including subsequent side reactions

Oxymyoglobin (MbO2) is oxidized easily to metmyoglobin (metMb) with generation of the superoxide anion, which can be converted by the spontaneous dismutation into H2O2, this being also a potent oxidant of MbO2. In the presence of sodium azide in stoichiometric amounts, however, the rate of autoxidation of MbO2 increased rapidly with increasing concentration of the anion, but soon reached a saturating level, the extent of which was about twice that of the normal autoxidation in buffer alone. Quantitative analysis has revealed that this enhancement is not due to the nucleophilic displacement of O2- from MbO2 by the anion (Satoh, Y., and Shikama, K. (1981) J. Biol. Chem. 256, 10272-10275), but is due to the additional oxidation of MbO2 by H2O2 freed from the metMb being occupied by the anion at the sixth coordination position. Based on these novel results and stoichiometric considerations, it is possible to propose a new view that H2O2 produced from O2- can be eliminated or decomposed mostly, if not completely, by the metMb resulting from the normal autoxidation reaction of MbO2, presumably via the formation of the ferryl species.     

Oxidation of oxymyoglobin to metmyoglobin with hydrogen peroxide: involvement of ferryl intermediate

Hydrogen peroxide, one of the potent oxidants in muscle tissues, can induce very rapid oxidation of oxymyoglobin (MbO2) to metmyoglobin (metMb) with an apparent rate constant of 7.5 X 10(4) h-1 M-1 (i.e., 20.8 s-1 M-1) over the wide pH range of 5.5-10.2 in 0.1 M buffer at 25 degrees C. Its molecular mechanism, however, is quite different from that of the autoxidation of MbO2 to metMb. Kinetic analysis has revealed that the hydrogen peroxide oxidation proceeds through the formation of ferryl-Mb(IV) from deoxy-Mb(II), which is in equilibrium with MbO2, by a two-equivalent oxidation with H2O2. Once the ferryl species is formed, it reacts rapidly with another deoxy-Mb(II) in a bimolecular fashion so as to yield 2 mol of metMb(III). Under physiological conditions, the rate-determining step was the oxidation of the deoxy species by H2O2, its rate constant being estimated to be on the order of 3.6 X 10(3) s-1 M-1 at 25 degrees C. These findings leads us to the view that a good supply of dioxygen provides rather an important defense against the oxidation of myoglobin with hydrogen peroxide in cardiac and skeletal muscle tissues.     

The interaction of oxymyoglobin with hydrogen peroxide: a kinetic anomaly at large excesses of hydrogen peroxide

The reaction of oxymyoglobin (MbO2) with H2O2 has been examined at pH 7.2 and 20(+/- 2) degrees C for reactant ratios of [H2O2]:[MbO2] greater than approximately 15:1. Under the conditions of large excesses of H2O2, the reaction is characterized by an increase in the rate of loss of MbO2 as [H2O2] is increased, for which a value of k(MbO2 + H2O2) approximately 3 M-1 s-1 is obtained. This kinetic behavior contrasts the saturation kinetics observed previously at lower values of [H2O2]. The change in kinetics at increasing excesses of H2O2 is accompanied by a progressive tendency toward the direct formation of ferrimyoglobin at the expense of ferrylmyoglobin formation. A mechanism is proposed in which an initially formed intermediate produces the ferryl derivative in competition with the formation of ferrimyoglobin through the interaction of further H2O2. Overall, the H2O2 is catalytically decomposed by the MbO2. This mechanism is integrated with that determined previously at low excesses of H2O2 into a complex general scheme that applies over the entire studied range of [H2O2]:[MbO2]. No evidence is obtained for the conversion of ferrylmyoglobin to oxymyoglobin by the large excesses of H2O2, regardless of whether the ferryl derivative is the product of the reaction of H2O2 with the oxy or ferri derivative of myoglobin.     

Study of electron transport in heme proteins. X. Effect of pH, ionic strength, and zinc ions and the rate of ferricytochrome c reduction by oxymyoglobin from swine heart]

The rate of the redox reaction between porcine MbO2 and ferri-Cyt c at different ionic strengths in the pH range 5-8 has been studied. At low ionic strength (I = 0-0.1) the pH dependence curve was found to have a sigmoid shape with pKeff approximately 5.7, implying the effect of ionization of His-119(GH1) at the "active site" of myoglobin on the kinetics of the process. In this range of ionic strengths the rate of the reaction decreases sharply. The slope of the curve in the coordinates of IgKexp versus square root of I/1 + square root of I varies depending on pH. It is greater at pH less than or equal to 6 and smaller at pH 7.5, which is due to deprotonation of His(GH1). At high ionic strength (I greater than 0.1) the rate of electron transfer is negligible, independent of pH and does not practically change as I increases from 0.1 to 1. It is shown that the local electrostatic interactions play a decisive role in the formation of an efficient electron-transfer complex between Mb and Cyt c. The binding of the zinc ion to His(GH1) was found to inhibit the electron transfer at I = 0.01, similarly to what occurs at a high ionic strength, though the "reactive" charges of the proteins are not screened and the positive charge at His(GH1) is retained. This suggests that His(GH1) is directly involved in the mechanism of electron transfer from Mb to Cyt c. The data obtained are compared with earlier data on the effect of pH, ionic strength and zinc ions on the reaction between MbO2 from sperm whale and Cyt c. To explain the higher efficiency of pig MbO2 as electron donor, the electrostatic and steric properties of both myoglobins have been analyzed.     

Electron transport in hemoproteins. IX. The effect of zinc ions on the rate of oxymyoglobin oxidation by ferricytochrome c

The effect of zink ions, which according to the X-ray data are bound to the His GH1 residue of myoglobin, has been investigated. It is shown that the electron transfer in the system is almost completely inhibited at the equimolar Zn2+ concentration in the pH range 5 to 8. Unlike the reaction between the intact MbO2 and Cyt c, the electron transfer rate in this case does not depend on pH and ionic strength of the solution. Further increase of Zn2+ concentration up to the 20-fold molar excess has no significant effect on the rate of the process. Since the thermodynamic characteristics of the redox reaction between MbO2 and Cyt c are not altered in the presence of Zn2+, the findings obtained can be interpreted as indicating the important role of His GH1 in the formation of productive electron transfer complex.     

High-pressure effect on the equilibrium and kinetics of cyanide binding to chloroperoxidase

The kinetics of cyanide binding to chloroperoxidase were studied using a high-pressure stopped-flow technique at 25 degrees C and pH 4.7 in a pressure range from 1 to 1000 bar. The activation volume change for the association reaction is delta V not equal to + = -2.5 +/- 0.5 ml/mol. The total reaction volume change, determined from the pressure dependence of the equilibrium constant, is delta V degrees = -17.8 +/- 1.3 ml/mol. The effect of temperature was studied at 1 bar yielding delta H not equal to + = 29 +/- 1 kJ/mol, delta S not equal to + = -58 +/- 4 J/mol per K. Equilibrium studies give delta H degrees = -41 +/- 3 kJ/mol and delta S degrees = -59 +/- 10 J/mol per K. Possible contributions to the binding process are discussed: changes in spin state, bond formation and conformation changes in the protein. An activation volume analog of the Hammond postulate is considered.     

The swinging movement of the distal histidine residue and the autoxidation reaction for midge larval hemoglobins.

Some insects have a globin exclusively in their fast-growing larval stage. This is the case in the 4th-instar larva of Tokunagayusurika akamusi, a common midge found in Japan. In the polymorphic hemoglobin comprised of 11 separable components, hemoglobin VII (Ta-VII Hb) was of particular interest. When its ferric met-form was exposed to pH 5.0 from 7.2, the distal histidine was found to swing away from the E7 position. As a result, the iron(III) was converted from a hexacoordinate to a pentacoordinate form by a concomitant loss of the axial water ligand. The corresponding spectral changes in the Soret band were therefore followed by stopped-flow and rapid-scan techniques, and the observed first-order rate constants of k(out) = 25 s(-1) and kin = 128 s(-1) were obtained for the outward and inward movements, respectively, of the distal histidine residue in 0.1 m buffer at 25 degrees C. For O2 affinity, Ta-VII Hb showed a value of P50 = 1.7 Torr at pH 7.4, accompanied with a remarkable Bohr effect (deltaH+ = -0.58) almost equal to that of mammalian hemoglobins. We have also investigated the stability property of Ta-VII HbO2 in terms of the autoxidation rate over a wide range of pH from 4 to 11. The resulting pH-dependence curve was compared with those of another component Ta-V HbO2 and sperm whale MbO2, and described based on a nucleophilic displacement mechanism. In light of the O2 binding affinity, Bohr effect and considerable stability of the bound O2 against acidic autoxidation, we conclude that T. akamusi Hb VII can play an important role in O2 transport and storage as the major component in the larval hemolymph.     

Kinetic study of the activation process of frog epidermis pro-tyrosinase by trypsin

1. The rate of tyrosinase formation has been calculated by coupling the activatory process of frog epidermis pro-tyrosinase by trypsin to the oxidation of L-DOPA to dopachrome. Under certain conditions ([trypsin]/[pro-tyrosinase] greater than or equal to 300), the lag period of the coupled reactions, tau, is independent of trypsin concentration. 2. The specific rate constant of tyrosinase formation at different temperatures has been calculated, ranging from 0.025 sec-1, at 5 degrees C to 0.248 sec-1, at 30 degrees C. 3. Thermodynamic parameters of the activatory process (delta G not equal to = + 18.5 kcal/mol; delta H not equal to = + 14.8 kcal/mol; delta S not equal to = -12.4 e.u.; Ea = + 15.3 kcal/mol), have been determined by the study of the system at different temperatures. These values are characteristic for a normal chemical reaction. 4. From these kinetic data, the order of products formation in the proteolytic step, can be determined, active tyrosinase being the last product released.     

Electron transfer in hemoproteins. VI. The dependence of the reduction rate of ferricytochrome c by oxymyoglobin on ionic strength

The rate of the redox-reaction between MbO2 and ferri-Cyt c has been investigated in the pH range 5-8 under different ionic strength of the solution. The influence of various anions-phosphate, chloride, sulfate and acetat on the rate of the reaction were also studied. It has been shown that under the low ionic strength, I less than 0.1, all pH-dependence curves have pronounced maximum near pH 6.0. While the ionic strength values increase in this interval the reaction rate falls markedly, the profile of lg k versus square root of I/1 + square root of I is linear. Under high ionic strength values, I greater than 0.1, the reaction rate in MbO2-Cyt c system is only slightly influenced by increasing salt concentrations and by pH changing. The results obtained support the idea that the local interactions of charged groups in " active sites" of MbO2 and Cyt c play the most important role in the mechanism of electron transfer. On the contrary net charges of the molecules have a negligible effect on the rate of the reaction. Compared to anions Cl-, SO42- and CH3COO- which influence the reaction rate in an analogous way, phosphate ions have essential inhibiting effect. This is most likely explained by the specific bonding of the phosphate ions to Cty c in the immediate vicinity from the site of the "active contact" with Mb molecule.     

A primitive myoglobin from Tetrahymena pyriformis: its heme environment, autoxidizability, and genomic DNA structure

A myoglobin-like protein isolated from Tetrahymena pyriformis is composed of 121 amino acid residues. This is much smaller than sperm whale myoglobin by 32 residues, suggesting a distinct origin from the common globin gene. We have therefore examined this unique protein for its structural, spectral and stability properties. As a result, the rate of autoxidation of Tetrahymena oxymyoglobin (MbO(2)) was found to be almost comparable to that of sperm whale MbO(2) over a wide range of pH 4-12 in 0.1 M buffer at 25 degrees C. Moreover, both pH profiles exhibited the remarkable proton-assisted process, which can be performed in sperm whale myoglobin by the distal (E7) histidine as its catalytic residue. These kinetic observations are also in full accord with spectral examinations for the presence of a distal histidine in ciliated protozoa myoglobin. At the same time, we have isolated the globin genes both from T. pyriformis and Tetrahymena thermophila, and found that there is no intron in their genomic structures. This is in sharp contrast to previous reports on the homologous globin genes from Paramecium caudatum and Chlamydomonas eugametos. Rather, the Tetrahymena genes seemed to be related to the cyanobacterial globin gene from Nostoc commune. These contracted or truncated globins thus have a marked diversity in the cDNA, protein, and genomic structures.     

Hydrogen peroxide plays a key role in the oxidation reaction of myoglobin by molecular oxygen. A computer simulation.

The stability properties of the iron(II)-dioxygen bond in myoglobin and hemoglobin are of particular importance, because both proteins are oxidized easily to the ferric met-form, which cannot be oxygenated and is therefore physiologically inactive. In this paper, we have formulated all the possible pathways leading to the oxidation of myoglobin to metmyoglobin with each required rate constant in 0.1 M buffer (pH 7.0) at 25 degrees C, and have set up six rate equations for the elementary processes going on in a simultaneous way. By using the Runge-Kutta method to solve these differential equations, the concentration progress curves were then displayed for all the reactive species involved. In this complex reaction, the primary event was the autoxidation of MbO2 to metMb with generation of the superoxide anion, this anion being converted immediately and almost completely into H2O2 by the spontaneous dismutation. Under air-saturated conditions (PO2 = 150 Torr), the H2O2 produced was decomposed mostly by the metMb resulting from the autoxidation of MbO2. At lower pressures of O2, however, H2O2 can act as the most potent oxidant of the deoxyMb, which increases with decreasing O2 pressures, so that there appeared a well defined maximum rate in the formation of metMb at approximately 5 Torr of oxygen. Such examinations with the aid of a computer provide us, for the first time, with a full picture of the oxidation reaction of myoglobin as a function of oxygen pressures. These results also seem to be of primary importance from a point of view of clinical biochemistry of the oxygen supply, as well as of pathophysiology of ischemia, in red muscles such as cardiac and skeletal muscle tissues.     

Aplysia oxymyoglobin with an unusual stability property

Native oxymyoglobin was isolated directly from the radular muscle of Aplysia kurodai with complete separation from metmyoglobin on a DEAE-cellulose column. It was examined for its spectral and stability properties. The spectrum of Aplysia MbO2 , which lacks the distal histidine, is very similar to those of mammalian oxymyoglobins , the alpha-peak being higher than the beta-peak and the absorbance ratio being 1.03. Its stability, however, is quite different from those of the mammalian oxymyoglobins , and Aplysia MbO2 is found to be extremely susceptible to autoxidation. Its rate is one-hundred times higher at pH 9.0, and its pH dependence is unusual and much less steep, when compared with sperm whale MbO2 as reference.     

High-pressure stopped-flow spectrometry at low temperatures

A stopped-flow instrument operating over temperature and pressure ranges of +30 to -20 degrees C and 10(-3) to 2 kbar , respectively, is described. The system has been designed so that it can be easily interfaced with many commercially available spectrophotometers of fast response time, with the aid of quartz fiber optics. The materials used for the construction are inert, metal free and the apparatus has proven to be leak free at temperatures as low as -20 degrees C under a pressure of 2 kbar . The performance of the instrument was tested by measuring the rate of reduction of cytochrome c with sodium dithionite and the 2,6-dichloroindophenol/ascorbate reaction. The dead time of the system has been evaluated to be 20, 50, and congruent to 100 ms in water at 20 degrees C, in 40% ethylene glycol/water, and at 20 degrees C and -15 degrees C, respectively. These values are rather pressure independent up to 2 kbar . Application of the bomb was demonstrated using the cytochrome c peroxidase/ethyl peroxide reaction. This process occurred in two phases and an increase in pressure decreased the rates of reactions indicating two positive volumes of activation (delta V not equal to app (fast) = 9.2 +/- 1.5 ml X mol-1; delta V not equal to app (slow) = 14 +/- 1.5 ml X mol-1, temperature 2 degrees C). The data suggest that the fast reaction could involve a hydrophobic bond, whereas the slow process could be associated with a stereochemical change of the protein. The problem of temperature equilibrium for high-pressure experiments is also discussed.     

Autoxidation of native oxymyoglobin. Kinetic analysis of the pH profile.

The rate of autoxidation of native oxymyoglobin to metmyoglobin has been examined over the pH range of 4.8--12.6 in 0.1 M buffer at 25 degrees C, and some 40 values of the observed first-order rate constant, kobs, are plotted against pH of the solution. In order to understand the kobs--pH profile thus obtained, some mechanistic models are proposed for the autoxidation reaction. The fitting of their rate equations as a function of pH has been examined to the experimental kobs-pH plot by a least-squares method with the use of a digital computer. The complicated pH-profile can be best explained by the 'acid-base catalyzed three states model', which reveals not only the catalytic role of hydrogen ions and hydroxyl ions, but also the involvement of two dissociation groups of myoglobin molecule in the autoxidation reaction.