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The rates of oxygen uptake by rat liver mitochondria (MC) (native coupled, freshly frozen, and uncoupled by FCCP) have been measured polarographically in the absence (V(0)) or presence (V(1)) of 0.11-0.25 mM sperm whale MbO2. Under the same standard conditions, the rate of sperm whale MbO2 deoxygenation (V(2)) has been studied spectrophotometrically in the presence of respiring MC. For freshly frozen MC, the dependence of V(1) and V(2) on the overall charge of MbO2 has been investigated at pH 5.6-7.6, and the influence of other differently charged proteins (apomyoglobin, egg lysozyme, lactalbumin, and BSA) has been studied at pH 7.4. It is shown that the rate of mitochondrial respiration in the presence of MbO2 increases by 10-30% (V(1) > V(0)). No myoglobin effect is observed for FCCP-uncoupled MC (V(max) does not change). The rate of MbO2 deoxygenation is equal to the rate of oxygen uptake by mitochondria (V(2)/V(1) ~ 1 at pH 7.2-7.5). At varying pH < 7.2, the V(2) values become markedly higher than V(1), evidently due to the increased MbO2 positive charge and its stronger interaction with negatively charged mitochondrial membrane. At pH 7.4, on the contrary, V(2) is twice lower than V(1) in the case of negatively charged CM-MbO2 (pI 5.2), which has carboxymethylated histidines. Positively charged lysozyme (pI 11) strongly inhibits MbO2 deoxygenation (V(2)) without affecting oxygen uptake by MC (V(0) and V(1)). At the same time, apomyoglobin (pI 8.5), which is structurally very similar to the holoprotein, and both negatively charged lactalbumin (pI 4.4) and BSA (pI 4.7) have no substantial influence on V(2) and V(1). The MC membrane evidently has no specific sites for the interaction with myoglobin. Rather, the protein contacts with phospholipids of the outer membrane during MbO2 deoxygenation, and electrostatic interactions are of great importance for this process.

In this review, we shortly summarize the data of our studies (and also corresponding studies of other authors) on the new mechanism of myoglobin (Mb) deoxygenation in a cell, according to which Mb acts as an oxygen transporter, and its affinity for the ligand, like in other transporting proteins, is regulated by the interaction with the target, in our case, mitochondria (Mch). We firstly found that contrary to previously formulated and commonly accepted concepts, oxymyoglobin (MbO) deoxygenation occurs only via interaction of the protein with respiring mitochondria (low p values are necessary but not sufficient for this process to proceed). Detailed studies of the mechanism of Mb-Mch interaction by various physicochemical methods using natural and artificial bilayer phospholipid membranes showed that: (i) the rate of MbO deoxygenation in the presence of respiring Mch fully coincides with the rate of O2 uptake by mitochondria from a solution irrespectively of their state (native coupled, freshly frozen, or FCCP-uncoupled), i.e. it is determined by the respiratory activity of Mch; (ii) Mb nonspecifically binds to membrane phospholipids of the outer mitochondrial membrane, while any Mb-specific protein or phospholipid sites on it are lacking; (iii) oxygen uptake by Mch from a solution and the uptake of Mb-bound oxygen are two different processes, as their rates are differently affected by proteins (e.g. lysozyme) that compete with MbO for binding to the mitochondrial membrane; (iv) electrostatic forces significantly contribute to the Mb-membrane interactions; the dependence of these interactions on ionic strength is provided by the local electrostatic interactions between anionic groups of phospholipids (the heads) and invariant Lys and Arg residues near the Mb heme pocket; (v) interactions of Mb with phospholipid membranes promote conformational changes in the protein, primarily in its heme pocket, without significant alterations in the protein secondary and tertiary structures; and (vi) Mb-membrane interactions lead to decrease in the affinity of myoglobin for O2, which could be monitored by the increase in the MbO autooxidation rate under aerobic conditions and under anaerobic ones, by the shift in the MbO/Mb(2) equilibrium towards the ligand-free protein. The decrease in the affinity of Mb for the ligand should facilitate O2 dissociation from MbO at physiological p values in cells.

In addition to reversible O2 binding, respiratory proteins of the globin family, hemoglobin (Hb) and myoglobin (Mb), participate in redox reactions with various metal complexes, including biologically significant ones, such as those of copper and iron. HbO and MbO are present in cells in large amounts and, as redox agents, can contribute to maintaining cell redox state and resisting oxidative stress. Divalent copper complexes with high redox potentials (E, 200-600 mV) and high stability constants, such as [Cu(phen)], [Cu(dmphen)], and CuDTA oxidize ferrous heme proteins by the simple outer-sphere electron transfer mechanism through overlapping π-orbitals of the heme and the copper complex. Weaker oxidants, such as Cu2+, CuEDTA, CuNTA, CuCit, CuATP, and CuHis (E ≤ 100-150 mV) react with HbO and MbO through preliminary binding to the protein with substitution of the metal ligands with protein groups and subsequent intramolecular electron transfer in the complex (the site-specific outer-sphere electron transfer mechanism). Oxidation of HbO and MbO by potassium ferricyanide and Fe(3) complexes with NTA, EDTA, CDTA, ATP, 2,3-DPG, citrate, and pyrophosphate PP proceeds mainly through the simple outer-sphere electron transfer mechanism via the exposed heme edge. According to Marcus theory, the rate of this reaction correlates with the difference in redox potentials of the reagents and their self-exchange rates. For charged reagents, the reaction may be preceded by their nonspecific binding to the protein due to electrostatic interactions. The reactions of LbO with carboxylate Fe complexes, unlike its reactions with ferricyanide, occur via the site-specific outer-sphere electron transfer mechanism, even though the same reagents oxidize structurally similar MbO and cytochrome b via the simple outer-sphere electron transfer mechanism. Of particular biological interest is HbO and MbO transformation into met-forms in the presence of small amounts of metal ions or complexes (catalysis), which, until recently, had been demonstrated only for copper compounds with intermediate redox potentials. The main contribution to the reaction rate comes from copper binding to the "inner" histidines, His97 (0.66 nm from the heme) that forms a hydrogen bond with the heme propionate COO group, and the distal His64. The affinity of both histidines for copper is much lower than that of the surface histidines residues, and they are inaccessible for modification with chemical reagents. However, it was found recently that the high-potential Fe(3) complex, potassium ferricyanide (400 mV), at a 5 to 20% of molar protein concentration can be an efficient catalyst of MbO oxidation into metMb. The catalytic process includes binding of ferrocyanide anion in the region of the His119 residue due to the presence there of a large positive local electrostatic potential and existence of a "pocket" formed by Lys16, Ala19, Asp20, and Arg118 that is sufficient to accommodate [Fe(CN)]. Fast, proton-assisted reoxidation of the bound ferrocyanide by oxygen (which i...

To determine the nature and characteristic parameters of the myoglobin-mitochondrion interaction during oxymyoglobin (MbO(2)) deoxygenation in the cell, we studied the quenching of the intrinsic mitochondrial flavin and tryptophan fluorescence by different liganded myoglobins in the pH range of 6-8, as well as the quenching of the fluorescence of the membrane probes 1,8-ANS and merocyanine 540 (M 540) embedded into the mitochondrial membrane. Physiologically active MbO(2) and oxidized metmyoglobin (metMb), which are unable to bind oxygen, were used as the quenchers. The absence of quenching of flavin and tryptophan fluorescence implies that myoglobin does not form quenching complexes with either electron transport chain proteins of the inner mitochondrial membrane or with outer membrane proteins. We found, however, that MbO(2) and metMb effectively quench 1,8-ANS and M 540 fluorescence in the pH range of 6-8. Characteristic parameters of 1,8-ANS and M 540 fluorescence quenching by the myoglobins (extent of quenching and quencher binding constant, K(m)) are very similar, indicating that both probes are localized in phospholipid sites of the mitochondrial membrane, and myoglobin is complexed with these sites. The dependence of K(m) on ionic strength proves the important role of coulombic interactions in the formation of the quenching complex. Since the overall charge of myoglobin is shown not to influence the K(m) values, the ionic strength dependence must be due to local electrostatic interactions in which polar groups of some part of the myoglobin molecule participate. The most likely candidates to interact with anionic groups of mitochondrial phospholipids are invariant lysine and arginine residues in the environment of the myoglobin heme cavity, which do not change their ionization state in the pH range investigated.