Ariki Matsuoka

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.

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.

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.

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.

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.

Aplysia myoglobins with an unusual amino acid sequence

The complete amino acid sequence of the myoglobin from Aplysia juliana, a species distributed world-wide, has been determined and compared with the sequence of the myoglobin of Aplysia limacina, a Mediterranean species, and of Aplysia kurodai, a Japanese and Asian species. Unlike mammalian myoglobins, Aplysia myoglobins contain only a single histidine residue, lacking the distal one, the homology being 76% between A. juliana and A. limacina, 74% between A. juliana and A. kurodai, and 83% between A. limacina and A. kurodai. The hydropathy profiles of the Aplysia myoglobins are very similar, but completely different from that of sperm whale myoglobin, taken as the reference.

Structure-Function Relationships in Unusual Nonvertebrate Globins

Based on the literature and our own results, this review summarizes the most recent state of nonvertebrate myoglobin (Mb) and hemoglobin (Hb) research, not as a general survey of the subject but as a case study. For this purpose, we have selected here four typical globins to discuss their unique structures and properties in detail. These include Aplysia myoglobin, which served as a prototype for the unusual globins lacking the distal histidine residue; midge larval hemoglobin showing a high degree of polymorphism; Tetrahymena hemoglobin evolved with a truncated structure; and yeast flavohemoglobin carrying an enigmatic two-domain structure. These proteins are not grouped by any common features other than the fact they have globin domains and heme groups. As a matter of course, various biochemical functions other than the conventional oxygen transport or storage have been proposed so far to these primitive or ancient hemoglobins or myoglobins, but the precise in vivo activity is still unclear. In this review, special emphasis is placed on the stability properties of the heme-bound O2. Whatever the possible roles of nonvertebrate myoglobins and hemoglobins may be (or might have been), the binding of molecular oxygen to iron(II) must be the primary event to manifest their physiological functions in vivo. However, the reversible and stable binding of O2 to iron(II) is not a simple process, since the oxygenated form of Mb or Hb is oxidized easily to its ferric met-form with the generation of superoxide anion. The metmyoglobin or methemoglobin thus produced cannot bind molecular oxygen and is therefore physiologically inactive. In this respect, protozoan ciliate myoglobin and yeast flavohemoglobin are of particular interest in their very unique structures. Indeed, both proteins have been found to have completely different strategies for overcoming many difficulties in the reversible and stable binding of molecular oxygen, as opposed to the irreversible oxidation of heme iron(II). Such comparative studies of the stability of MbO2 or HbO2 are of primary importance, not only for a full understanding of the globin evolution, but also for planning new molecular designs for synthetic oxygen carriers that may be able to function in aqueous solution and at physiological temperature.

Relationship between oxygen affinity and autoxidation of myoglobin.

Studies using myoglobins reconstituted with a variety of chemically modified heme cofactors revealed that the oxygen affinity and autoxidation reaction rate of the proteins are highly correlated to each other, both decreasing with decreasing the electron density of the heme iron atom. An Fe(3+)-O(2)(-)-like species has been expected for the Fe(2+)-O(2) bond in the protein, and the electron density of the heme iron atom influences the resonance process between the two forms. A shift of the resonance toward the Fe(2+)-O(2) form results in lowering of the O(2) affinity due to an increase in the O(2) dissociation rate. On the other hand, a shift of the resonance toward the Fe(3+)-O(2)(-)-like species results in acceleration of the autoxidation through increasing H(+) affinity of the bound ligand.

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.