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.

Native oxymyoglobin (Mb02) 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 MbOz used as a reference, Paramecium MbO, was found to be much more susceptible to autoxidation over a wide range of pH (4-11) in 0.1 M buffer at 25°C. Kinetic analysis has revealed that a proton-catalyzed displacement of 0; from M b 0 2 by an entering water molecule can play a dominant role in the autoxidation reaction of Paramecium Mb02 to metMb, as in the case of sperm whale MbOz involving the distal histidine as its catalytic residue. At pH values higher than 9.5, however, Paramecium MbOz 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 [Iwaasa, H. et al. (1989) J . Mol. Biol. 208,.The occurrence of a hemoglobin-like protein in protozoa was first reported by Sat0 and Tamiya on the basis of spectroscopic observations on cell suspensions of Paramecium caudatum [I]. This was later confirmed by Keilin and Ryley with estimations of its content of 1.12 -1.74% [2]. Isolation and characterization of the so-called Paramecium hemoglobin, which is functionally referred to rather as myoglobin [3], have thus been carried out from Paramecium aurelia [4], P. tetraurelia [3], and P. caudatum and P. primaurelia [5].Very recently, we have determined the complete amino acid sequence of a myoglobin isolated from P. caudatum [6]. It consists of 116 amino acid residues with a molecular mass of 12565 Da including the heme moiety, this being much smaller than mammalian myoglobins by 37 residues. The protein contains two histidine residues at positions 68 and 84, but we failed to find any notable degree of sequence similarity with other known hemoglobins. In relation to these structural studies, the stability properties of the oxygenated form of Paramecium myoglobin should be of most importance and of primary concern, but these are little understood.In this paper, we describe procedures for isolating oxymyoglobin directly from the ciliated cells of P. caudatum and examine for the first time the autoxidation rate of oxymyoglobin (MbO,) to metmyoglobin (metMb) over a wide range of pH values. The resulting pH dependence was subjected to some mechanistic analysis in order to unravel the elementary processes involved in the stability properties of Paramecium MbOz. This kinetic analysis seems to be of great interest when compared with that of sperm whale MbOz as a reference, and should deepen our insight into the myoglobinhemoglobin chemistry and the evolution of these molecules from protozoa to higher animals, since the kinetics of the autoxidation should reflect differences on the distal side of the heme iron [7, 81. Correspondence to K. Shikama, Biological Institute,...