A comparison of the respiratory chain in particles from Paracoccus denitrificans and bovine heart mitochondria by EPR spectroscopy

A study is presented on the EPR characteristics of the paramagnetic groups in the respiratory chain present in membrane particles of Paracoccus denitrificans, the respiratory system of which is very similar to that in submitochondrial particles from beef heart. All paramagnetic prosthetic groups of the mitochondrial system are also found in the bacterial plasma membrane. Their properties suggest that the respiratory groups are embedded in very similar protein environments in the two systems.     

The role of iron-sulfur center 2 in electron transport and energy conservation in the NADH-ubiquinone segment of the respiratory chain in Paracoccus denitrificans.

1. The electron paramagnetic resonance spectra at 15 K of reduced membrane particles of Paracoccus denitrificans exhibit resonance signals with g values, line shapes and temperature profile which are similar to the signals of the iron-sulfur centers observed in the NADH-ubiquinone segment of mitochondrial respiratory chains. These iron-sulfur centers are reducible with NADH, NADPH as well as chemically with dithionite. 2. Sulphate-limited growth of Paracoccus denitrificans results in the loss of an electron paramagnetic resonance signal (gz approximately 2.05, gy approximately gx approximately 1.92) which has properties similar to those of iron-sulfur center 2 of the NADH dehydrogenase of mitochondrial origin. The loss of this signal is accompanied by a decrease in the NADH oxidase and NADH ferricyanide oxidoreductase activities to respectively 30 and 40% of the values found for succinate-limited growth conditions. In addition respiration in membrane particles from sulphate-limited cells loses its sensitivity to rotenone. 3. Since sulphate-limited growth of Paracoccus denitrificans induces loss of site I phosphorylation [Arch. Microbiol. (1977) 112, 25-34] these observations suggest a close correlation between site I phosphorylation, rotenone-sensitivity and the presence of an electron paramagnetic resonance signal with gz approximately 2.05 and gy approximately gx approximately 1.92.     

The Role of Reactive Oxygen Species in Mitochondrial Permeability Transition

We have provided evidence that mitochondrial membrane permeability transition induced by inorganic phosphate, uncouplers or prooxidants such as t-butyl hydroperoxide and diamide is caused by a Ca²⁺-stimulated production of reactive oxygen species (ROS) by the respiratory chain, at the level of the coenzyme Q. The ROS attack to membrane protein thiols produces cross-linkage reactions, that may open membrane pores upon Ca²⁺ binding. Studies with submitochondrial particles have demonstrated that the binding of Ca²⁺ to these particles (possibly to cardiolipin) induces lipid lateral phase separation detected by electron paramagnetic resonance experiments exploying stearic acids spin labels. This condition leads to a disorganization of respiratory chain components, favoring ROS production and consequent protein and lipid oxidation.     

Anaerobic respiration and energy conservation in Paracoccus denitrificans. Functioning of iron-sulfur centers and the uncoupling effect of nitrite.

1. Electron paramagnetic resonance spectra at 8-60 K of NADH-reduced membrane particles prepared from Paracoccus denitrificans grown anaerobically with nitrate as terminal electron acceptor show the presence of iron-sulfur centers 1-4 in the NADH-ubiquinone segment of the respiratory chain. In addition resonance lines at g = 2.058, g = 1.953 and g = 1.88 are detectable in the spectra of succinate-reduced membranes at 15 K, which are attributed to the iron-sulfur-containing nitrate reductase. 2. Sulphate-limited growth under anaerobic conditions does not affect the iron-sulfur pattern of NADH dehydrogenase or nitrate reductase. Furthermore respiratory chain-linked electron transport and its inhibition by rotenone are not influenced. These results contrast those observed for sulphate-limited growth of P. denitrificans under aerobic conditions [Eur. J. Biochem. (1977) 81, 267-275]. 3. Proton translocation studies of whole cells indicate that nitrite increases the proton conductance of the cytoplasmic membrane, resulting in a collapse of the proton gradient across the membrane. Nitrite accumulates under anaerobic growth conditions with nitrate as terminal electron acceptor; the extent of accumulation depends on the specific growth conditions. Thus the low efficiencies of respiratory chain-linked energy conservation observed during nitrate respiration [Arch. Microbiol. (1977) 112, 17-23] can be explained by the uncoupling action of nitrite.     

EPR spectroscopic analysis of binding sites of a cancerostatic agent on erythrocyte membranes

A biologically highly active antitumor agent, chloroethyl-nitrosourea-piperidine-N-oxyl, was found to bind covalently to sulfhydryl groups of membrane proteins of erythrocytes. The agent penetrates the erythrocyte membrane very easily within three minutes. Mainly two different binding sites are distinguishable with rather high rotational mobility of the NO-moiety and high polarity of their environment. The lowered broadening of bound label suggests pocket-like binding sites with a limited accessibility for paramagnetic ions. Besides common concepts that antitumor agents often bind to DNA and inhibit cell proliferation, further targets on plasma membranes are taken into consideration for antitumor agents.     

The orientation of iron-sulfur clusters and a spin-coupled ubiquinone pair in the mitochondrial membrane.

Oriented multilayers made from beef heart and yeast mitochondria and submitochondrial particles were studied using electron paramagnetic resonance. EPR signals from membrane-bound iron-sulfur clusters and from a spin-coupled ubiquinone pair are highly orientation dependent, implying that these redox centers are fixed in the membrane at definite angles relative to the membrane plane. Typically the iron-iron axis (gz) of the binuclear iron-sulfur clusters is in the membrane plane. This finding is discussed in terms of the protein structure. The tetranuclear iron-sulfur clusters can have their gz axis either perpendicular or parallel to the membrane plane, but intermediate orientation was not observed.     

The transcytosis of divalent metal transporter 1 and apo-transferrin during iron uptake in intestinal epithelium

Caco-2 cells grown in bicameral chambers are a model system to study intestinal iron absorption. Caco-2 cells exhibit constitutive transport of iron from the apical (luminal) chamber to the basal (serosal) chamber that is enhanced by apo-transferrin in the basal chamber, with the apo-transferrin undergoing endocytosis to the apical portion of the cell. With the addition of iron to the apical surface, divalent metal transporter 1 (DMT1) on the brush-border membrane (BBM) undergoes endocytosis. These findings suggest that in Caco-2 cells DMT1 and apo-transferrin may cooperate in iron transport through transcytosis. To prove this hypothesis, we determined by confocal microscopy that, after addition of iron to the apical chamber, DMT1 from the BBM and Texas red apo-transferrin from the basal chamber colocalized in a perinuclear compartment. Colocalization was also observed by isolating endosomes from Caco-2 cells after ingestion of ultra-small paramagnetic particles from either the basal or apical chamber. The isolated endosomes contained both transferrin and DMT1 independent of the chamber from which the paramagnetic particles were endocytosed. These findings suggest that iron transport across intestinal epithelia may be mediated by transcytosis.     

Effects of ethanol and acetaldehyde on iron-sulfure centers in the mitochondrial respiratory chain.

Incubation of submitochondrial particles with relatively low concentrations of ethanol (20–100 mm) or acetaldehyde (1–10 mm) produces alterations in the electron paramagnetic resonance spectra of the iron-sulfur centers in the NADH dehydrogenase segments of the respiratory chain. The iron-sulfur centers in the NADH dehydrogenase region are most sensitive to both ethanol and acetaldehyde, in comparison to the iron-sulfur centers in succinate dehydrogenase and the cytochrome b-c region. Centers N-3, 4, N-5, 6 and N-1b are affected after relatively short incubation periods (3–30 min) while center N-2 shows considerable sensitivity over somewhat longer incubations (20–90 min). The most ethanol-sensitive center in the succinate dehydrogenase region of the respiratory chain is high potential iron-sulfur protein-type center S-3. Potentiometric analysis shows that these alterations are not due to simple changes in the redox state caused by addition of dissolved oxygen. Changes in the electron paramagnetic resonance spectra can be correlated with decreased rates of oxidation of NADH and, to a lesser extent, succinate in both ethanol- and acetaldehyde-treated submitochondrial particles.     

Paramagnetic probes of multicomponent electron-transfer systems

Electron-transfer systems associated with steroid hormone production in the adrenals and with photosynthetic NADP+ reduction in the chloroplast share some important features. Complexes between components in both systems affect the exposure of prosthetic groups to water-soluble paramagnetic groups. The sensitivity of the components to probes of different charges suggests that electrostatic interactions are important in complex formation. Haem plane orientation in cytochrome P-450 of the adrenal system is parallel to the membrane plane in both native and reconstituted systems.     

Identification and transmembranous localization of active cytochrome oxidase in reconstituted membranes of purified phospholipids by electron microscopy

Cytochrome oxidase vesicles with high oxidase activity and respiratory control ratio (greater than 3.5) were characterized by the freeze-etch technique for electron microscopy. By the use of this technique, cytochrome oxidase is shown to be an inner membrane particle. By locating cross-fractured vesicles in the same preparation, cytochrome oxidase particles are shown to extend across the phospholipid bilayer membranes. When cytochrome oxidase is added to preformed liposomes respiratory control is not observed, but high oxidase activity is maintained. In this preparation the cytochrome oxidase particles are located on the outer vesicle membrane surface. These observations provide direct evidence that cytochrome oxidase is found in a transmembranous position in closed, activecytochrome oxidase vesicles having respiratory control.     

Adrenal chromaffin granules: evidence for an ultrastructural equivalent of the proton-pumping ATPase

Adrenal chromaffin granules are known to possess an F1-ATPase which according to biochemical criteria is very similar to the mitochondrial one. To find a morphological equivalent for this enzyme chromaffin granules from bovine adrenal medullar were subjected to negative staining and freeze-etching. With both methods globular particles of 8 to 9 min diameter could be demonstrated on the surface of these organelles. A single granule possessed on average 22 particles. In negative staining the particles appeared separated from the membrane by a stalk of 8 nm. This typical morphological appearance was independent from a great variety of experimental procedures. After freeze-etching the particles were closely apposed to the membrane without any evidence for an interposed stalk. Pretreatment of chromaffin granules with pronase or trypsin led to a time dependent disappearance of the surface particles. In negative staining the stalked of chromaffin granules were found to be very similar in structure and size to those of mitochondria which have already been identified as F1-complexes. Based on this observation and other lines of evidence we suggest that the stalk particles found on the surface of chromaffin granules represent the F1-complex of the proton-pumping ATPase of these organelles.     

The use of coated paramagnetic particles as a physical label in a magneto-immunoassay

An ideal label for use in an immunoassay would require no further chemical or electromagnetic stimulation prior to its detection and would be free from interference from the sample matrix. Micron sized paramagnetic particles are able to perturb magnetic fields. This perturbation can be directly detected using a suitable electronic device and is independent of the sample matrix. In this study coated paramagnetic particles were used as a physical label in a non-competitive solid phase ‘sandwich’ assay for the detection of human transferrin. The transferrin acted as a ‘biological bridge’ allowing a dose dependant immobilization of the paramagnetic particles to a polyethylene terephthalate solid phase. Quantitation of the paramagnetic label was achieved using an electronic detection system allowing a linear dose response with a femtomolar detection limit (260 fmol).     

Intramitochondrial positions of ubiquinone and iron-sulphur centres determined by dipolar interactions with paramagnetic ions.

E.p.r. (electron-paramagnetic-resonance) spectra of ubisemiquinone (QH) organic radicals and all of the known iron-sulphur centres were studied in normal and 'nickle-plated' pigeon heart mitochondria, submitochondrial particles and submitochondrial particles from which succinate dehydrogenase had been removed. Incubation of pigeon heart mitochondria, submitochondrial particles or succinate dehydrogenase-depleted submitochondrial particles with substrate in the presence of pure O2 results in the accumulation of Q-H. In mitochondria, the e.p.r. spectrum of Q-H is characterized by in-homogeneous line broadening. A heterogeneous population of semiquinones appears to be partly responsible for these effects in mitochondria. Additon of Ni(II) to mitochondria renders saturation of the Q-H resonance more difficult. On the other hand, the resonance in either submitochondrial particles or succinate dehydrogenase-depleted particles is narrower than the same spectrum in mitochondria, and saturates like a homogeneous line. The presence of Ni(II) in either of these preparations, further, has no effect on either the A-H spectrum or the saturation curve. Therefore QH appears to be situated on the exterior surface of the mitochondrion. Likewise, the e.p.r. spectra and saturation curves of iron-sulphur centre N-2 exhibit characteristics of inhomogeneous line broadening, not only in intact mitochondria but also in both submitochondrial particles and succinate dehydrogenase-depleted particles. Because of the small pool size of centre N-2, this effect is likely to arise from a spin interaction with some other component in the membrane. Ni(II) has no effect on the saturation in centre N-2 in mitochondria or submitochondrial particles, and only a marginal effect in the succinate dehydrogenase-depleted preparation. These results are indeterminate with respect to the position of centre N-2 in the membrane; but suggest that its distance from the succinate dehydrogenase binding site is on the order of 1 nm. All of the other ferredoxin-type iron-sulphur centres in both preparations were not affected by paramagnetic ions. Homogeneous e.p.r. spectra and saturation curves are observed for both of the HiPIP-type (high-potential iron-sulphur protein-type) iron-sulphur centres in mitochondrial centres S-3 and bc-3. Addition of No(II) to intact mitochondria results in a dipolar interaction with centre bc-3. No effect was observed on centre S-3 in either preparation. A comprehensive model is presented for the structure of the respiratory electron-transport system in mitochondria, based on e.p.r. relaxation studies in the present and the preceding paper. There is no direct evidence for transmembrane electron flow through any of the known energy-coupling sites in mitochondria, so that direct hydrogen atom transfer across the membrane (as a combination of H+ translocation coupled to electron flow) does not occur...     

Current-voltage relationships for proton flow through the F0 sector of the ATP-synthase, carbonylcyanide-p-trifluoromethoxyphenylhydrazone or leak pathways in submitochondrial particles

Respiring submitochondrial particles from which the F1 sector of ATP-synthase was displaced generated a membrane potential in the range of 115-140 mV. Addition of oligomycin raised the membrane potential by approximately 40 mV. The lower membrane potential in particles with F1 displaced is attributed to partial dissipation of the proton electrochemical gradient as a consequence of proton flow through the open proton channels provided by the F0 sectors of the ATP-synthase. The characteristics of proton flow through the open F0 channels were studied by varying the rate of electron transport-driven proton translocation which permitted the establishment of a range of steady-state membrane potentials. Open F0 channels appeared to have a gated response to the membrane potential such that they were inoperative when the potential fell below approximately 110 mV. The membrane potential was measured as a function of respiratory rate in intact Mg-ATP submitochondrial particles that had been treated with low concentrations of the protonophore carbonylcyanide-p-trifluoromethoxyphenylhydrazone. In general a linear dependence of membrane potential upon respiratory rate was observed except at the lowest concentrations of protonophore and highest respiratory rates, presumably because the effect of the protonophore was then offset by an increased rate of proton translocation driven by the respiratory chain. The effect of increasing concentrations of carbonylcyanide-p-trifluoromethoxyphenylhydrazone on the membrane potential of respiring submitochondrial particles was studied. It was found that equal amounts of the protonophore lowered the membrane potential to a lesser extent at lower values of the membrane potential. Treatment of Mg-ATP submitochondrial particles with oligomycin slightly increased (by approximately 10 mV) the size of the respiration-dependent membrane potential, but did not alter the profile of membrane potential as a function of succinate oxidation rate. The latter was controlled by titration with malonate. This result indicates that the F0 sector of the ATP-synthase does not significantly contribute to leak pathways in intact submitochondrial particles.     

The use of ferromagnetic Dacron as solid-phase in chemiluminescent assays

Ferromagnetic Dacron hydrazide particles (63–74 m) were activated either by converting hydrazide groups to azide or binding glutaraldehyde to the hydrazide groups. Human serum albumin (HSA) was immobilised to the activated matrices and antibodies against HSA were detected by using the chemiluminescent assay. The use of these particles (either Dacron-azide or Dacron-glutaraldehyde) gave similar results compared with the paramagnetic particles commercially available from Ciba Corning.     

Lipid-protein interactions in human plasma LDL evidenced by magnetic resonance

Low density lipoprotein (LDL) particles exhibit extremely complex three-dimensional structural organization which is still not understood at the molecular level. The aim of this study was to provide the experimental evidence of a direct non-covalent interaction of the protein part with the lipid matrix. The approach was based on the combination of (1)H NMR (600 MHz) spectroscopy with thiol-specific spin labeling of the protein (apoB). It is shown that the spectral peaks assigned to the methyl head groups of phosphatidylcholine and sphingomyelin in the (1)H spectra of LDL exhibit line broadening when otherwise free thiol groups of apoB are covalently modified by methanethiosulfonate spin label. The effect is similar in the presence of water soluble paramagnetic compound. These results indicate that fragments of apoB, which are part of the receptor binding region, are directly in contact with the solvated phospholipid head groups of the lipid matrix.     

Ferritin as a label for high-gradient magnetic separation.

In three model systems, particles the size of cells or smaller have been surface labeled with ferritin to make them slightly paramagnetic, by virtue of the iron in the ferritin. In each case it was possible to show that labeled particles could be magnetically removed from a flowing suspension by the high-gradient magnetic separation (HGMS) technique. The first system of particles consisted of small (1 micron) carboxylate-modified latex spheres to which ferritin was covalently bound to create stable paramagnetic particles analogous to a ferritin-labeled subcellular membrane preparation. In the second system polyacrylamide beads that more closely approximated whole cells in size (5-50 microns) were labeled with immunoferritin. The third system was a biomembrane preparation: erythrocyte ghosts labeled with a ferritin-lectin conjugate. A field of 7 T (tesla) (70 kG) was used in each case, along with buffer flow rates through the HGMS column in the range 0.1-1.0 ml/min.     

Two different pathways for necrotic cell death induced by free radicals

Plasma membrane modifications have been widely recognized as crucial factors in cell injury and death. One of these modifications, surface blebbing, has been considered as an injury-marker associated with a series of biochemical and physiological modifications. Our study focused on the different effects of free radical-induced cell damage by quinone menadione (2-methyl-1,4-naphthoquinone) and by hyperthermic shock (45°C) on the erythroleukemic cell line K.562. Different techniques including immunofluorescence, freeze-fracturing, and electron paramagnetic resonance spectroscopy were employed. Menadione induced the formation of surface blebs, accompanied by a rearrangement of the microfilament system and changes in the distribution of plasma membrane proteins. In contrast, heat-shocked cells showed neither blebbing nor important cytoskeletal changes. Finally, the electron paramagnetic resonance results showed an increase in membrane order not specifically related to the type of free radical-induced stress. These cell death features appear to suggest the existence of two different types ofpathways for necrotic cell death: both treatments induce cell injury and eventual death by modifiting plasma membrane integrity and function. However, one involves cytoskeleton-dependent surface blebbing, whereas the other does not.Abbreviations EPR electron paramagnetic resonance - HS heat shock - IMPs intramembrane particles - MEN menadione     

Erythrocyte spectrin maintains its segmental motions on oxidation: a spin-label EPR study.

The segmental motions of cross-linked erythrocyte skeletal protein (spectrin-actin-protein 4.1) samples, labeled with nitroxide spin labels, were monitored by conventional first-harmonic and saturation transfer second-harmonic electron paramagnetic resonance methods. Skeletal proteins were extracted from human red blood cells and treated with three oxidative reagents (diamide, hydrogen peroxide, and phenylhydrazine) to cross-link sulfhydryl groups and with one fixative reagent (glutaraldehyde) to cross-link lysine residues. The treatments provided extensive cross-linking between spectrin-actin-protein 4.1 molecules, as determined by gel electrophoresis, and surface charge modification, as determined by pl measurements. However, segmental motions of the cross-linked skeletal proteins remained generally similar to those in normal skeletal proteins. Both the weakly immobilized and the strongly immobilized motions were similar in cross-linked and control samples. Small differences in some motional components were detected. In some cases, faster mobilities were observed, with approximately 5% of the strongly immobilized motions converted to the weakly immobilized motions in the cross-linked samples. It is often believed that the consequence of membrane protein oxidation is restricted protein dynamics, giving membrane rigidity. However, our studies provide needed experimental evidence to indicate that segmental motions are maintained with very little modification even in the presence of extensive cross-linking. Thus cross-linking does not restrict the internal molecular flexibility that gives rise to segmental motions.     

Membranes of bacteria and mechanism of action of the antibiotic gramicidin S]

The cyclopeptide antibiotic gramicidin S taken at a concentration of 100--200 mkg/mg membrane protein rapidly increases the permeability of M. lysodeikticus protoplast membranes for substrates of respiratory chain and exogenous cytochromes c. Prolonged incubation of gramicidin S with protoplasts results in their lysis which is more fast at low temperatures. In contrast to natural gramicidin, a derivative of gramicidin S with acetylated amino groups does not inhibit either the micrococcus membrane dehydrogenase or the whole of respiratory chain and does not affect the osmotic barrier of protoplasts. Aliphatic diamines (at concentrations up to 0.1 M) and Ca2+ ions (10(-2) M) do not affect the functioning of the respiratory chain in isolated micrococcus membranes. Another derivative of the antibiotic with an increased distance of loaded amino groups from the cyclopeptide framework (diglycyl gramicidin S) affects the membrane in a way similar to that of natural gramicidin. Washing of gramicidin-treated membranes with NaCl enhances the inhibitory effect of the antibiotic on membrane enzymes. The data obtained suggest that in addition to ionic interactions some hydrophobic interactions also occur during gramicidin S binding to the bacterial membrane, probably at the expense of a hydrophobic peptide ring. It is assumed that gramicidin S, similar to Ca2+ and some other membranotropic agents provides for phase separation of negatively charged phospholipids from other groups of phospholipids, manifesting itself in an appearance of "frozen" sites on the membrane which destroys its barrier properties. This is due to the formation of ionic bonds of negatively charged phospholipids. Simultaneously, unlike Ca2+, gramicidin S, when interacting with membrane proteins, prevents their redistribution in more liquid parts of the membrane, which results in a situation when the respiratory enzymes become surrounded by alkyl chains with restricted motion.