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.

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.

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.

1H NMR study of dynamics and thermodynamics of acid-alkaline transition in ferric hemoglobin of a midge larva (Tokunagayusurika akamusi).

One of the components of hemoglobin from the larval hemolyph of Tokunagayusurika akamusi possesses naturally occurring substitution at the E7 helical position (Leu E7) [M. Fukuda, T. Takagi, K. Shikama, Biochim. Biophys. Acta 1157 (1993) 185-191]. Its oxygen affinity is almost comparable to those of mammalian myoglobins and it exhibits Bohr effect. Both acidic and alkaline forms of the ferric hemoglobin have been investigated using 1H NMR in order to gain insight into molecular mechanisms for relatively high oxygen affinity and Bohr effect of this protein. The NMR data indicated that the acidic form of the protein possesses pentacoordinated heme, and that the alkaline form possessing OH- appears with increasing the pH value. pH titration yielded a pK value of 7.2 for the acid-alkaline transition, and this value is the lowest among the values reported so far for various myoglobins and hemoglobins. The kinetic measurements of the transition revealed that the activation energy for the dissociation of the Fe-bound OH-, as well as the dissociation and association rates, decrease with increasing the pH value. These pH dependence properties are likely to be related to the Bohr effect of this protein.

The molecular mechanism of autoxidation for human oxyhemoglobin. Tilting of the distal histidine causes nonequivalent oxidation in the beta chain.

Human oxyhemoglobin showed a biphasic autoxidation curve containing two rate constants, i.e. kf for the fast autoxidation due to the alpha chains, and ks for the slow autoxidation of the beta chains, respectively. Consequently, the autoxidation of the HbO2 tetramer produces two different curves from the pH dependence of kf and ks. The analysis of these curves revealed that the beta chain of the HbO2 tetramer does not exhibit any proton-catalyzed autoxidation, unlike the alpha chain, where a proton-catalyzed process involving the distal histidine residue can play a dominant role in the autoxidation rate. When the alpha and beta chains were separated from the HbO2 tetramer, however, each chain was oxidized much more rapidly than in the tetrameric parent. Moreover, the separated beta chain was recovered completely to strong acid catalysis in its autoxidation rate. These new findings lead us to conclude that the formation of the alpha1beta1 contact produces in the beta chain a conformational constraint whereby the distal histidine at position 63 is tilted away slightly from the bound dioxygen, preventing the proton-catalyzed displacement of O-2 by a solvent water molecule. The beta chains have thus acquired a delayed autoxidation in the HbO2 tetramer.

Biphasic nature in the autoxidation reaction of human oxyhemoglobin.

In comparison with myoglobin molecule as a reference, we have studied the autoxidation rate of human oxyhemoglobin (HbO2) as a function of its concentration in 0.1 M buffer at 35 degrees C and in the presence of 1 mM EDTA. At pH 6.5, HbA showed a biphasic autoxidation reaction that can be described completely by a first-order rate equation containing two rate constants-kf, for fast autoxidation of the alpha-chain, and ks, for slow autoxidation of the beta-chain, respectively. When tetrameric HbO2 was dissociated into alpha beta-dimers by dilution, the value of kf increased markedly to an extent comparable with the autoxidation rate of horse heart oxymyoglobin (MbO2). The rate constant Ks, on the other hand, was found to remain at an almost constant value over the whole concentration range from 1.0 x 10(-3) M to 3.2 x 10(-6) M in heme. At pH 8.5 and pH 10.0, however, the autoxidation of HbO2 was monophasic, and no enhancement in the rate was observed by diluting hemoglobin solutions. Taking into consideration the effects of 2,3-diphosphoglyceric acid and chloride anion on the autoxidation rate of HbO2, we have characterized the differential susceptibility of the alpha- and beta-chains to the autoxidation reaction in aqueous solution.

The unique structures of protozoan myoglobin and yeast hemoglobin: an evolutionary diversity.

A hemoglobin-like protein is found in some of the single-celled organisms, but its structure is quite different from that of mammalian myoglobin or hemoglobin. For instance, a protozoan myoglobin isolated from Paramecium caudatum consists of 116 amino acid residues, so that this contracted form is nearly two thirds of sperm whale myoglobin. Yeast hemoglobin from Candida norvegensis, on the other hand, is composed of a single polypeptide chain with 387 amino acid residues, but of two distinct domains carrying different functions; that is the N-terminal, heme-containing region and the C-terminal, FAD-containing reductase domain. The very unique structures of these ancient hemoproteins tell us their own strategies to overcome many difficulties in the reversible and stable binding of molecular oxygen, a very strong oxidizing agent, to the heme iron(II) in aqueous solutions.

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.

The structure of the Aplysia kurodai gene encoding ADP-ribosyl cyclase, a second-messenger enzyme.

The complete nucleotide (nt) sequences of the cDNA and gene encoding the marine mollusk Aplysia kurodai (Ak) ADP-ribosyl cyclase (ADRC) which synthesizes cyclic ADP-ribose (cADP-ribose), a second messenger for Ca2+ mobilization from endoplasmic reticulum, were determined. Ak ADRC consists of 258 amino acids (aa) (29 kDa). It shares 86% aa sequence homology with that from A. californica, and 31-32% homology with the human, rat and mouse cluster of differentiation 38 (CD38) that has both ADRC and cADP-ribose hydrolase activities. The Ak ADRC-encoding gene (ADRC) spans approx. 7 kb and contains eight exons and seven introns. The transcription start point (tsp) determined by primer extension analysis and S1 mapping is 28 bp downstream from the TATA box. This gene is expressed specifically in the ovotestis, although the mammalian CD38-encoding gene is expressed in many kinds of tissues and cells. The 5'-flanking region contains several consensus sequences responsible for the germ-cell-specific expression of the mouse zona pellucida 3 (ZP3) and Drosophila melanogaster chorion genes. The existence of the consensus sequences located at nt -1649, -1161, -234 and -90 may account for the ovotestis-specific expression of the Ak ADRC gene.

The pH-dependent swinging-out of the distal histidine residue in ferric hemoglobin of a midge larva (Tokunagayusurika akamusi).

Hemoglobin VII (TaVII) is a major component in the larval hemolymph of Tokunagayusurika akamusi, a common midge (Diptera) found in Japan. This protein contains 150 amino-acid residues including the usual distal histidine at position 64. When the aquomet-form was placed in acidic pH range, its Soret peak was considerably blue-shifted and accompanied by a marked decrease in intensity, indicative of the protein being converted into a structure quite similar to that of Aplysia myoglobin lacking the distal histidine residue. The pH-dependent magnetic circular dichroism spectra in the Soret region have also revealed that TaVII hemoglobin is in an equilibrium between a hexacoordinate and a pentacoordinate structure for its ferric heme iron. We attribute this to a transition from an iron-ligated water molecule that is hydrogen-bonded to the distal histidine, to a water-free iron with the histidine swung-out of the heme pocket. Furthermore, this process was described by the involvement of a single dissociable group with pKa = 6.3 in 0.1 M KCl at 25 degrees C.

Polymorphic hemoglobin from a midge larva (Tokunagayusurika akamusi) can be divided into two different types.

The hemoglobin from the 4th-instar larva of Tokunagayusurika akamusi, a common midge found in eutrophic lakes in Japan, was composed of as many as 11 separable components (IA, IB, II, III, IV, V, VIA, VIB, VII, VIII, IX) on a DEAE-cellulose column. However, we have found that these components can be divided into two groups on the basis of their spectroscopic properties, one being named as the normal type (N-type) and the other being referred to as the low type (L-type). Since the major difference between them seemed to be the presence or absence of the distal (E7) histidine residue, which plays an important role in the stability properties of the bound dioxygen, the complete amino acid sequence was then determined for each typical component, namely, VII (N-type) and V (L-type): the former hemoglobin contained the usual distal histidine residue at position 64, whereas the latter one replaced it by isoleucine at position 66. The homology test for 40 N-terminal amino acid residues of all components also demonstrates that T. akamusi hemoglobin is composed of two different clusters showing a very early separation in the phylogenetic tree.

Primary structure of Tetrahymena hemoglobins.

The primary structure of hemoglobins of Tetrahymena thermophila and T. pyriformis was determined by peptide and cDNA sequence analysis. Their sequences were 83.5% identical to each other and homologous to other protozoan and cyanobacteria hemoglobins, but not to proteins of the globin family. An intron was not present in the T. thermophila hemoglobin gene.

Decomposition of hydrogen peroxide by metmyoglobin: a cyclic formation of the ferryl intermediate.

1. Metmyoglobin reacted with H2O2 to form ferryl-myoglobin, which reverted back spontaneously to the met-form. 2. Through this cyclic reaction of myoglobin between metMb(III) and ferryl-Mb(IV), we proposed that H2O2, one of the potent oxidants in vivo, can be decomposed continuously in cardiac and skeletal muscle tissues in the absence of catalase and peroxidase.

The Soret magnetic circular dichroism of ferric high-spin myoglobins. A probe for the distal histidine residue.

To find a simple criterion for the presence of the distal (E7) histidine residue in myoglobins and hemoglobins, the Soret magnetic-circular-dichroic spectra were examined for ferric metmyoglobins from various species. A distinct and symmetric dispersion-type curve was obtained for myoglobins containing the distal histidine, whereas a relatively weak and unsymmetric pattern was observed for myoglobins lacking this residue, such as those from three kinds of gastropodic sea molluscs, a shark and the African elephant. The magnetic-circular-dichroic spectra obtained would thus be a direct reflection of the presence or absence of a water molecule at the sixth coordinate position of the heme iron(III), this axial water ligand being stabilized by hydrogen-bond formation to the distal histidine residue. On the basis of these Soret magnetic-circular-dichroic signals, we also examined the structure of a protozoan myoglobin (or a monomeric hemoglobin) from Paramecium caudatum of particular interest for the evolution of these proteins from protozoa to higher animals.

Amino acid sequence of yeast hemoglobin. A two-domain structure.

The complete amino acid sequence of a hemoglobin from yeast (Candida norvegensis) has been determined by peptide and cDNA sequence analyses. The protein is composed of 387 amino acid residues and its amino terminus was blocked by an acetyl group. A computer search showed that the sequence of 155 N-terminal residues has 39% homology with that of Vitreoscilla hemoglobin. On the other hand, the sequence of 230 C-terminal residues showed a small, but notable, degree of similarity with that of a methemoglobin reductase found in human erythrocyte, i.e. NADH-cytochrome b5 oxido-reductase. We therefore conclude that yeast hemoglobin consists of two distinct domains; one is a heme-containing oxygen binding domain of the N-terminal region and the other is an FAD-containing reductase domain found in the C-terminal region.

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.

Stability properties of Aplysia oxymyoglobin: effect of esterification of the heme propionates.

When compared with sperm whale MbO2 as a reference Aplysia MbO2 is extremely susceptible to autoxidation, and its pH dependence for the reaction is also unusual. Kinetic and thermodynamic analyses have shown that two types of carboxyl group with pKa = 4.3 and 6.1 at 25 degrees C are involved in the stability property of Aplysia MbO2, the protonation of these groups being responsible for an increase in its autoxidation rate in the acidic pH range. Since protoheme has two carboxyl groups of the propionic acid side-chains, we have prepared Aplysia myoglobin containing the dimethylester of protohemin. The autoxidation of this derivative was found to be described by the involvement of only one type of carboxyl group with pKa = 4.4. A possible candidate for the protein residue was therefore discussed on the basis of the known amino acid sequences of three Aplysia myoglobins: it is suggested that Glu-94, next to the proximal His-95, is the most likely candidate.

Autoxidation of oxymyoglobin: a meeting point of the stabilization and the activation of molecular oxygen.

1. The primary events of haemoprotein reactions with molecular oxygen have been re-examined by placing special emphasis upon the reduction properties of dioxygen. 2. In the stepwise reduction of O2 to water via hydrogen peroxide, the addition of the first electron is an unfavourable, uphill process with the midpoint potential of -0.33 V, all the subsequent steps being downhill. This thermodynamic barrier to the first step is, therefore, a most crucial ridge located between the stabilization and the activation of dioxygen performed by haemoproteins. 3. If the proteins have a redox potential much higher than -0.33 V, molecular oxygen must bind to the proteins stably and reversibly. In Mb or Hb, however, the FeO2 centre is always subject to a nucleophilic attack of the water molecule or hydroxyl ion, which can enter the haem pocket from the surrounding solvent. These can cause irreversible oxidation of the FeO2 bonding to the ferric met-form with generation of the superoxide anion. 4. In cases of the oxygen activation, if haemoproteins have a redox potential lower than or close to -0.33 V, the first reduction of O2 to O2- would be a spontaneous process. Cytochrome P-450 provides such an example and can facilitate the subsequent addition of electrons that leads to the breaking of the O-O bond to yield the hydroxylating species. 5. As to the proteins whose redox potential is not facilitative and appreciably higher than -0.33 V, a bimetallic, concerted, two-equivalent reduction of the bound dioxygen to the peroxide level would be much more favoured without the intermediate formation of O2-. This is probably the case of cytochrome c oxidase for the reduction of O2 to water. 6. The redox potential diagrams thus visualize various aspects of the ways haemoproteins overcome their thermodynamic constraints and carry out their specific functions in the stabilization and the activation of molecular oxygen.

Protozoan myoglobin from Paramecium caudatum. Its autoxidation reaction and hemichrome formation.

Native oxymyoglobin (MbO2) was isolated directly from the cells of Paramecium caudatum with complete separation from metmyoglobin (metMb) on a DEAE-cellulose column. It was examined for its spectral and stability properties. When compared with sperm whale MbO2 used as a reference, Paramecium MbO2 was found to be much more susceptible to autoxidation over a wide range of pH (4-11) in 0.1 M buffer at 25 degrees C. Kinetic analysis has revealed that a proton-catalyzed displacement of O2- from MbO2 by an entering water molecule can play a dominant role in the autoxidation reaction of Paramecium MbO2 to metMb, as in the case of sperm whale MbO2 involving the distal histidine as its catalytic residue. At pH values higher than 9.5, however, Paramecium MbO2 was found to be oxidized to yield a hemichrome. The spontaneous formation of hemichromes is at variance with the other known myoglobins and is therefore discussed in relation to the unusual amino acid sequence of Paramecium myoglobin having a large number of deletion.