Keiji Shikama

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. k f for the fast autoxidation due to the α chains, andk s for the slow autoxidation of the β chains, respectively. Consequently, the autoxidation of the HbO2tetramer produces two different curves from the pH dependence ofk f and k s . The analysis of these curves revealed that the β chain of the HbO2tetramer does not exhibit any proton-catalyzed autoxidation, unlike the α chain, where a proton-catalyzed process involving the distal histidine residue can play a dominant role in the autoxidation rate. When the α and β chains were separated from the HbO2tetramer, however, each chain was oxidized much more rapidly than in the tetrameric parent. Moreover, the separated β chain was recovered completely to strong acid catalysis in its autoxidation rate. These new findings lead us to conclude that the formation of the α1β1 contact produces in the β 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 β chains have thus acquired a delayed autoxidation in the HbO2 tetramer.

Autoxidation of native oxymyoglobin from bovine heart muscle

A method is described for the preparation of native oxymyoglobin from bovine heart muscle. The aqueous extract is gel filtered on Sephadex G-50 to isolate myoglobin from hemoglobin. Native oxymyoglobin is then separated from metmyoglobin by DEAE-cellulose chromatography. There is a marked effect of temperature on the autoxidation of native oxymyoglobin to metmyoglobin, with Q10 values approximating 5.3 over the pH range of 5–10. The activation energies over this pH range are shown to be almost constant, i.e., 26.5 kcal·mole−1. In contrast to the suggestions in earlier reports, the autoxidation rate of native oxymyoglobin estimated at physiological pH and temperature is quite high with t12 ≤ 1.5 days under air saturation. This suggests the existence of an in vivo system(s) immediately reducing metmyoglobin formed to the ferrous state.

Protozoan myoglobin from Paramecium caudatum: Its unusual amino acid sequence

A protozoan myoglobin (or monomeric hemoglobin) was isolated from Paramecium caudatum, and its complete amino acid sequence determined. It consists of 116 amino acid residues with a molecular mass of 12,565 daltons, this being much smaller than sperm whale myoglobin by 37 residues and even smaller than a bacterial hemoglobin from Vitreoscilla by 30 residues in terms of the monomer unit. A computer search showed no notable sequence homology with other hemoproteins. It contains two histidine residues at positions 68 and 84, but other lines of evidence seem to be needed to complete their final alignment. This is the first protozoan myoglobin to be sequenced. It may provide us with a new molecular basis for a further understanding of myoglobin-hemoglobin chemistry and their evolution.

Stability properties of dioxygen-iron(II) porphyrins: an overview from simple complexes to myoglobin

Abstract The reversible and stable binding of molecular oxygen to iron(II) is not a simple process. Although it has been possible to synthesize a class of O 2 -binding porphyrins by the introduction of certain steric restraints to prevent the formation of an oxygen-bridged dimer, small heme complexes are mostly oxidized very rapidly and irreversibly by O 2 . In cases of myoglobin and hemoglobin, each heme iron atom is embedded in its protein matrix so as to be unable to form such a dimer. Nevertheless, the oxygenated form is still oxidized to the ferric met-species, at a slow but considerable rate, with the generation of superoxide anion. Kinetic and thermodynamic studies of the stability of native oxymyoglobin have revealed that its autoxidation proceeds through a pathway quite different from that of the simple heme complexes. The FeO 2 center of myoglobin is always subject to nucleophilic attack by an entering water molecule with strong proton assistance from the distal histidine (E 7), which acts as a catalytic residue. This center is also open to the attack of an entering hydroxide anion. These reactions can cause irreversible displacement of the bound dioxygen from MbO 2 in the form of O 2 − so that the iron is converted to the ferric met-form. Myoglobin has thus evolved with a globin moiety that can protect the FeO 2 center from easy access of a water molecule and its conjugate anionic species. These new features of the stability of MbO 2 are of primary importance, not only for a full understanding of the nature of FeO 2 bonding, but also for planning new molecular designs for synthetic oxygen carriers which may be able to function in aqueous solutions under physiological conditions.

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.

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.

Spectral properties unique to the myoglobins lacking the usual distal histidine residue

Myoglobins can be divided into two groups. One group contains the usual myoglobins that have histidine at the distal (E7) position, and the other contains a few, but interesting myoglobins that lack the usual distal histidine residue. Spectroscopic examinations have shown that there is a remarkable difference in the Soret band between the two types of myoglobin, and an absorbance ratio of the Soret peak of the acidic met-form to that of the oxy-form seems to be very useful as a simple criterion for predicting whether or not a myoglobin has the usual distal histidine residue.

Amino acid sequence of myoglobin from Aplysia kurodai

Abstract The complete amino acid sequence of the myoglobin from Aplysia kurodai, a common gastropodic mollusc on the Japanese coast, has been determined. The myoglobin is composed of 144 amino acid residues, is acetylated at the amino terminus and contains one histidine residue at position 95. It is also of interest that the myoglobin differs in amino acid sequence from that of Aplysia limacina, a Mediterranean species, by 21 replacements and one deletion.

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.

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.