Myoglobin and mitochondria: oxymyoglobin interacts with mitochondrial membrane during deoxygenation.

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.

[Myoglobin and mitochondria: kinetics of oxymyoglobin deoxygenation in mitochondria suspension].

The kinetics of whale MbO2 deoxygenation was studied spectrophotometrically in the presence of breathing rat mitochondria under conditions when mitochondria were separated from the protein solution by a semipermeable film capable to transfer only low-molecular-weight compounds and directly in the solution of MbO2 with mitochondria (incubation medium: 15-35 mM succinate, 150 mM sucrose, 100 mM KCl, 0.5 mM EGTA, 5 mM KH2PO4, 10 mM MOPS, pH 7.4). It was shown that the splitting of O2 from MbO2 at physiological pO2 is possible only if it directly contacts mitohondria. The deoxygenation rate does not depend on the protein concentration (zero order on [MbO2] as opposite to the first order reaction in the absence of mitochondria) and completely coincides with the rate of oxygen consumption by mitochondria under the same conditions, as indicated by the polarographic data. The dependence of the MbO2 deoxygenation rate on the concentration of mitochondria and the protein, and on the total charge of the MbO2 molecule was studied using horse MbO2 (pI 7.1), sperm whale MbO2 (pI 8.3), its zinc complex, Zn-MbO2 (pI > 8.3), and the sperm whale MbO2 derivative carboxymethylated at His residues, CM-MbO2 (pI 5.2). The mechanism of MbO2 deoxygenation in the cell obviously actuates its interplay with the mitochondrial membrane. As a result, the affinity of Mb to oxygen decreases several times, which corresponds to a shift of the Mb dissociation curve to higher pO2 values.

Myoglobin content in skeletal muscles of hibernating ground squirrels rises in autumn and winter.

The content of myoglobin (Mb) in skeletal muscles of Arctic Yakutian ground squirrel (Citellus undulatus Pallas) was measured in the active euthermic summer and prehibernating autumn animals as well as in hibernating and awake animals in winter. The myoglobin content in winter, irrespective of the state of the animal, was found to be about three times higher than in summer. The content of myoglobin in autumn was also two-fold increased compared to summer, suggesting that high myoglobin level is necessary for hibernation. Analysis of biochemical data available suggests that the increase in myoglobin content in winter is probably related to a high oxygen demand of muscles at the first stage of arousal (non-shivering thermogenesis) when rectal temperature rises from 0 to 10-12 degrees C. At this stage, the oxygen-dependent processes in muscles proceed under the conditions when peripheral blood flow is blocked and anaerobic glycolysis is switched off.

[The myoglobin level in skeletal muscles of the hibernating Iakutiia ground squirrel Citellus undulatus]. / Izucheniie soderzhaniia mioglobina v skeletnoi muskulature zimnespiashchego iakutskogo suslika Citellus undulatus.

Many characteristics of the ground squirrel metmyoglobin (Mb) were found to be similar to those of the pig and different from the whale ones. The Mb content in winter time was 2.5-3.0-fold higher in hibernating and arousing ground squirrels than in alert ones. This seems to be connected with the high demand of oxygen in the muscles at the first stage of arousal.

Seasonal changes in myoglobin content in muscles of hibernating Yakutian ground squirrels.

Myoglobin content in skeletal muscles of the Yakutian ground squirrel Citellus undulatus was measured during different periods of the annual life cycle: in active animals in summer, and in hibernating and awake animals in winter. It was found that the Mb content in winter, irrespective of the state of the animal (hibernating or awake), is 2.5-3-fold higher than in summer. Analysis of biochemical data available in the literature suggests that the increase in Mb content in winter is most probably related to a high oxygen demand of muscles at the first stage of arousal when the body temperature rises from 0 to 10-12 degrees C (non-shivering thermogenesis or thermoregulatory tonus). At this stage, the oxygen-dependent processes in muscles proceed under conditions of blocked peripheral blood flow and failure of anaerobic glycolysis.

[Study of squirrel myoglobin. Oxidation-reduction reaction between squirrel oxymyoglobin and ferricytochrome c]. / Izuchenie mioglobina suslika. Okislitel'no-vosstanovitel'naia reaktsiia mezhdu oksimioglobinom suslika i ferritsitokhromom c.

Metmyoglobin (met-Mb) of Yakutian ground squirrel (Citellus undulatus) was isolated from skeletal muscles and fractionated using gel and ion-exchange chromatography. An electrophoretically homogeneous major fraction of met-Mb (pI 7.05) was obtained with a 90% yield. The rates of the redox reaction between ground squirrel oxy-Mb and horse and ground squirrel ferri-Cyt c were studied in the pH range of 5-8 at ionic strengths ranging from 0.01 to 1. The experimental results were compared to the earlier obtained data for sperm whale and pig Mbs whose three-dimensional structures and physico-chemical properties are well studied. It was shown that the properties of ground squirrel Mb are very similar to those of pig Mb but differ from those of whale Mb. The amino acid sequences of about 50 Mbs from various mammals as well as from some avian and chordate species (Protein Data Bank) were compared. It was found that rodent myoglobins can be divided into two distinct groups. Myoglobins of animals living in water are identical with those of sperm whale and other cetaceans, while myoglobins of ground rodents closely resemble pig Mb. The number of non-homologous amino acid substitutes in each of the two groups (1-2 positions) is much less compared to all rodent myoglobins (8 positions). The charge structure of the Mb contact site interacting with Cyt c in the course of the electron transfer reaction is strictly conservative for all mammalian Mbs tested in this study (but not for birds and chordates). It is assumed that under certain extreme conditions oxy-Mb can directly interact with cell organelles, such as mitochondria.

[Study of electron transport in heme proteins. X. Effect of pH, ionic strength, and zinc ions and the rate of ferricytochrome c reduction by oxymyoglobin from swine heart]. / Izuchenie perenosa élektrona v gemoproteidakh. X. Vliianie pH, ionnoi sily i ionov tsinka na skoros' vosstanovleniia ferritsitokhroma c oksimioglobinom iz serdtsa svin'i.

The rate of the redox reaction between porcine MbO2 and ferri-Cyt c at different ionic strengths in the pH range 5-8 has been studied. At low ionic strength (I = 0-0.1) the pH dependence curve was found to have a sigmoid shape with pKeff approximately 5.7, implying the effect of ionization of His-119(GH1) at the "active site" of myoglobin on the kinetics of the process. In this range of ionic strengths the rate of the reaction decreases sharply. The slope of the curve in the coordinates of IgKexp versus square root of I/1 + square root of I varies depending on pH. It is greater at pH less than or equal to 6 and smaller at pH 7.5, which is due to deprotonation of His(GH1). At high ionic strength (I greater than 0.1) the rate of electron transfer is negligible, independent of pH and does not practically change as I increases from 0.1 to 1. It is shown that the local electrostatic interactions play a decisive role in the formation of an efficient electron-transfer complex between Mb and Cyt c. The binding of the zinc ion to His(GH1) was found to inhibit the electron transfer at I = 0.01, similarly to what occurs at a high ionic strength, though the "reactive" charges of the proteins are not screened and the positive charge at His(GH1) is retained. This suggests that His(GH1) is directly involved in the mechanism of electron transfer from Mb to Cyt c. The data obtained are compared with earlier data on the effect of pH, ionic strength and zinc ions on the reaction between MbO2 from sperm whale and Cyt c. To explain the higher efficiency of pig MbO2 as electron donor, the electrostatic and steric properties of both myoglobins have been analyzed.

[Electron transport in hemoproteins. IX. The effect of zinc ions on the rate of oxymyoglobin oxidation by ferricytochrome c]. / Izuchenie perenosa élektrona v gemoproteinakh. IX. Vliianie ionov tsinka na skorost' okisleniia oksimioglobina ferritsitokhromom c.

The effect of zink ions, which according to the X-ray data are bound to the His GH1 residue of myoglobin, has been investigated. It is shown that the electron transfer in the system is almost completely inhibited at the equimolar Zn2+ concentration in the pH range 5 to 8. Unlike the reaction between the intact MbO2 and Cyt c, the electron transfer rate in this case does not depend on pH and ionic strength of the solution. Further increase of Zn2+ concentration up to the 20-fold molar excess has no significant effect on the rate of the process. Since the thermodynamic characteristics of the redox reaction between MbO2 and Cyt c are not altered in the presence of Zn2+, the findings obtained can be interpreted as indicating the important role of His GH1 in the formation of productive electron transfer complex.

[Binding of bis-(2-guanidoethyl)disulfide with DNA and polyriboadenylic acid in an aqueous solution. Study by ultrasonic velocimetry]. / Sviazyvanie bis-(2-guanidoétil)disul'fida s DNK i poliriboadenilovoikislotoi v vodnom rastvore. Issledovanie metodom ul'trazvukovoi velosimetrii.

The interaction of DNA and poly(A) with bis-(2-guanidoethyl)disulfide (GED) which is the most simple model of arginine containing proteins has been investigated by means of ultrasonic velocimetry. It has been shown that the association of GED with nucleic acids proceeds by two stages. The stoichiometric relationships and association constants for each of these stages are estimated. A mechanism of the binding is proposed.

[Reversal of B to A transition in DNA by bis-(20guanidoethyl)disulfide]. / Obrashchenie B--A-perekhoda v DNK bis(2-guanidoetil)disul'fidom.

Transition from B to A form of DNA in water/ethanol solutions is hindered by stoichiometric quantities of a bifunctional cation, bis-(2-guanidoethyl)disulfide (GED). Using the data on the shift and widening of the B--A transition curves in the presence of GED, a value of the order of 10 base pairs has been estimated for the cooperativity length of the B--A transition which agrees well with the values earlier obtained.

[Peculiarities of DNA winding with bifunctional cations]. / Osobennosti zakruchivaniia DNK bifunktsional'nymi kationamn.

Addition of the bifunctional cations, bis(2-aminoethyl) disulphide-cystamine, and bis(2-guanidoethyl) disulphide-GED, into water solution of DNA results in a decrease in magnitude of the positive circular dichroism (CD) band. However, unlike the similar effect due to alcaline ions or ions of ammonium and guanidinium the above effect occurs at much smaller, stoichiometric with phosphates, concentrations of the dications. Another peculiarity of GED is the attaining of a plato region for the curve of the CD change with the rise of GED concentration. Since the decrease in positive band CD is connected with increase in the rotation angle between the base pairs, the observed behavior of the bifunctional cations are supposedly due to peculiarities of their winding action upon DNA helix. Reducing the disulphide bond in the cations gives rise to increase in the DNA positive CD band up to the values inherent to those in the presence of the corresponding monocations. The higher efficiency of the bifunctional cations is thus due to the cationic groups belong to one and the same molecule. Such compounds could thus be considered as a simple model of DNA-protein interaction.

[Effect of low-molecular amines on DNA conformation and stability of the double helix]. / Vliianie nizhomolekuliarnykh aminov na konformatsiiu i stabil'nost' dvoinoi spirali DNK

Interaction of low-molecular amines (cystamine, cysteamine, cystaphose, asparagine, beta-alanine) with DNA was studied. The amines change the positive circular dichroism (CD) band of DNA as well as temperature and range width of melting. Effect of amines on DNA depends on ionic strength of the solvent, concentration and structure of the ligand. Monamines cause destabilization of DNA double helix followed by stabilization as ligand concentration increases. At concentrations stabilizing the double helix DNA conformation undergoes transition from the B- to C-form. The results obtained enable to relate the stabilizing effect of low-molecular amines and conformational B leads to C-transition to the non-specific interaction of ligand amino groups with DNA phosphates, and the destabilizing effect of monoamines of low concentrations to their interaction with bases, mainly in the denaturated sites of DNA. It is proposed that a stronger effectiveness of amines as compared to monovalent metals in the conformational shift of DNA towards the C-form is due to the additional effect of disturbance of hydrophobic interactions in DNA double helix.