Yoshiaki Sugawara

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.

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.

Aplysia myoglobin with an unusual heme environment

Abstract Unlike mammalian myoglobins, Aplysia myoglobin contains a single histidine residue that most likely corresponds to the heme-binding proximal one, its oxygenated form is extremely unstable and its CD magnitude is about two-thirds that of sperm whale myoglobin. A structural prediction also indicates that Aplysia myoglobin has an unusual heme environment distinctly altered from that of the mammalian myoglobins.