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Exogenous and endogenous cytochrome c

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... Two axial ligands, His18 and Met80, are coordinated to the heme iron of cytochrome c. Met80 readily dissociates from the heme iron in oxidized cytochrome c, and instead exogenous ligands such as cyanide ion, azide ion, imidazole, pyridine, and various other anions have been reported to coordinate to the heme iron [3][4][5][6][7]. At alkaline pH, Met80 of cytochrome c has been shown to dissociate from the heme iron and instead Lys72, Lys73, or Lys79 coordinates to the heme iron [8][9][10][11]. ...
... Cyanide ion forms a stable complex with the ferric heme iron in heme proteins, allowing it to be used as a probe of the active site character. Cyanide ion has been shown to bind to oxidized cytochrome c at relatively high concentration (approximately 50 mM) [5,22]. Binding of cyanide ion to cytochrome c is sensitive to the stability/flexibility of its heme region [23], and cyanide ion has been reported to bind to the alkaline isomer and acid-induced conformation of cytochrome c [24,25]. ...
... The Q band at 529 nm of oxidized dimeric cytochrome c redshifted to 536 nm on addition of cyanide ion (Fig. S1). Although it is known that cyanide ion binds to monomeric cytochrome c at a high cyanide ion concentration of about 50 mM, causing similar redshifts in the Soret and Q bands [3,5,6], no significant change was observed in the absorption spectrum of monomeric cytochrome c on addition of 1 mM cyanide ion (Fig. S2). Difference absorption spectra were calculated by subtracting the spectrum of dimeric cytochrome c in the absence of cyanide ion from the spectra of dimeric cytochrome c in the presence of cyanide ion. ...
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We have previously shown that methionine–heme iron coordination is perturbed in domain-swapped dimeric horse cytochrome c. To gain insight into the effect of methionine dissociation in dimeric cytochrome c, we investigated its interaction with cyanide ion. We found that the Soret and Q bands of oxidized dimeric cytochrome c at 406.5 and 529 nm redshift to 413 and 536 nm, respectively, on addition of 1 mM cyanide ion. The binding constant of dimeric cytochrome c and cyanide ion was obtained as 2.5 × 104 M−1. The Fe–CN and C–N stretching (ν Fe–CN and ν CN) resonance Raman bands of CN−-bound dimeric cytochrome c were observed at 443 and 2,126 cm−1, respectively. The ν Fe–CN frequency of dimeric cytochrome c was relatively low compared with that of other CN−-bound heme proteins, and a relatively strong coupling between the Fe–C–N bending and porphyrin vibrations was observed in the 350–450-cm−1 region. The low ν Fe–CN frequency suggests weaker binding of the cyanide ion to dimeric cytochrome c compared with other heme proteins possessing a distal heme cavity. Although the secondary structure of dimeric cytochrome c did not change on addition of cyanide ion according to circular dichroism measurements, the dimer dissociation rate at 45 °C increased from (8.9 ± 0.7) × 10−6 to (3.8 ± 0.2) × 10−5 s−1, with a decrease of about 2 °C in its dissociation temperature obtained with differential scanning calorimetry. The results show that diatomic ligands may bind to the heme iron of dimeric cytochrome c and affect its stability.
... It is here suggested that the difficulty may lie in differences in the properties of exogenous and endogenous cytochrome c, similar to those already pointed out by TSOU'S experiments (10). Wainio et al. (11) recently stated that they had succeeded in demonstrating this reaction, but their results show some inconsistencies that need to be explained in detail: their data clearly show that the 100 PM ferrocytochrome c that were added became about 80 per cent oxidized during an experiment which was presumably carried out in the complete absence of oxygen. ...
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The ubiquinol-2 or duroquinol oxidoreductase activity of mitochondrial ubiquinol-cytochrome c oxidoreductase was titrated with combinations of antimycin, myxothiazol and N,N'-dicyclohexylcarbodiimide (DCCD). A statistical model has been developed that can predict the activity of the complex treated with all possible combinations of these inhibitors. On the basis of the measured titration curves the model had to accommodate interaction between the two promoters of the complex. The titrations confirm that treatment with DCCD results in the modification of a certain site in one of the two promoters of the bc1 dimer, thereby blocking one antimycin A binding site without inhibiting electron transfer. Modification of both antimycin A binding sites of the dimer is apparently required for inhibition of electron transfer through the complex, just as modification of both myxothiazol-binding sites is required for full inhibition. The conclusion can be drawn that mitochondrial ubiquinol-cytochrome c oxidoreductase is a functional dimer, consisting of electrically interacting protomers.
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Reconstitution of respiratory control and the energy-dependent pyridine nucleotide transhydrogenation linked to succinate oxidation by soluble succinate dehydrogenase and the dehydrogenase-depleted particles is demonstrated. The restoration of succinate oxidation and respiratory control is a function of the concentration of succinate dehydrogenase present. Externally added succinate dehydrogenase effects neither the rate nor the respiratory control of NADH oxidation. The results are discussed in terms of the structural organization of the respiratory components in the mitochondrial membrane.
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Rebinding dynamics of CO to carboxymethyl cytochrome c (Ccytc), a chemically modified cytochrome c to bind ligands in its ferrous form, in D(2)O solution at 283 K after photodeligation, was investigated using femtosecond vibrational spectroscopy. The stretching mode of CO bound to the protein shows four stretching bands near 1962 cm(-1). Time-resolved spectra of the bound CO revealed a slight band-position-dependent rebinding kinetics, suggesting that the geminate rebinding of CO depends on the conformation of the protein. The overall rebinding kinetics of CO to Ccytc was more than 1000 times faster than that to myoglobin (Mb), a ligand-binding protein, and is also faster than a model heme, microperoxidase-8 in viscous solvent. The efficient rebinding of CO to Ccytc was attributed to the longer retention of the dissociated CO near the active binding site by the organized protein matrix of Ccytc. The spectra of the dissociated CO reveal a fast-growing band in the picosecond time scale that is assigned to CO in D(2)O solvent. The ultrafast CO escape to bulk solution is consistent with its 3D structure showing a sizable opening in the active site. It appears that most of the dissociated CO rebinds within 1 ns, except for those that escape to the bulk solution through the opening. The CO rebinding in Ccytc indicates that the primary heme pocket in Mb, located near the active site and holding the dissociated ligand for longer than tens of nanoseconds, has a specific structure to suppress CO rebinding.
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1. By the application of the principle of the sequential fragmentation of the respiratory chain, a simple-method has been developed for the isolation of phospholipid-depleted and phospholipid-rich cytochrome oxidase preparations. 2. The phospholip-rich oxidase contains about 20% lipid, including mainly phosphatidylethanolamine, phosphatidylcholine, and cardiolipin. Its enzymic activity is not stimulated by an external lipid such as asolectin. 3. The phospholipid-depleted oxidase contains less than 0.1% lipid. It is enzymically inactive in catalyzing the oxidation of reduced cytochrome c by molecular oxygen. This activity can be fully restored by asolectin; and partially restored (approximately 75%) by purified phospholipids individually or in combination. The activity can be partially restored also by phospholipid mixtures isolated from mitochondria, from the oxidase itself, and from related preparations. Among the detergents tested only Emasol-1130 and Tween 80 show some stimulatory activity. 4. The phospholipid-depleted oxidase binds with cytochrome c evidently by "protein-protein" interactions as does the phospholipid-rich or the phospholipid-replenished oxidase to form a complex with the ratio of cytochrome c to heme a of unity. The complex prepared from phospholipid-depleted cytochrome oxidase exhibits a characteristic Soret absorption maximum at 415 nm in the difference spectrum of the carbon monoxide-reacted reduced form minus the reduced form. This 415-nm maximum is abolished by the replenishment of the complex with a phospholipid or by the dissociation of the complex in cholate or in a medium of high ionic strength. When ascorbate is used as an electron donor, the complex prepared from phospholipid-depleted cytochrome oxidase does not cause the reduction of cytochrome a3 which is in dramatic contrast to the complex from the phospholipid-rich or the phospholipid-replenished oxidase. However, dithionite reduces cytochrome a3 in all of the preparations of the cytochrome c-cytochrome oxidase complex. These facts suggest that the action of phospholipid on the electron transfer in cytochrome oxidase may be at the step between cytochromes a and a3. This conclusion is substantiated by preliminary kinetic results that the electron transfer from cytochrome a to a3 is much slower in the phospholipid-depleted than in phospholipid-rich or phospholipid-replenished oxidase. On the basis of the cytochrome c content, the enzymic activity has been found to be about 10 times higher in the system with the complex (in the presence of the replenishedhe external medium unless energy is provided, and that
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1. Beef heart mitochondria have a cytochrome c1:c:aa3 ratio of 0.65:1.0:1.0 as isolated; Keilin-Hartree submitochondrial particles ahve a ratio of 0.65:0.4:1.0. More than 50% of the submitochondrial particle membrane is in the 'inverted' configuration, shielding the catalytically active cytochrome c. The 'endogenous' cytochrome c of particles turns over at a maximal rate between 450 and 550 s-1 during the oxidation of succinate or ascorbate plus TMPD; the maximal turnover rate for cytochrome c in mitochondria is 300-400 s-1, at 28 degrees-30 degrees C, pH 7.4. 2. Ascorbate plus N,N,N',N'-tetramethyl-p-phenylene diamine added to antimycin-treated particles induces anomalous absorption increases between 555 and 565 nm during the aerobic steady state, which disappear upon anaerobiosis; succinate addition abolishes this cycle and permits the partial resolution of cytochrome c1 and cytochrome c steady states at 552.5-547 nm and 550-556.5 nm, respectively. 3. Cytochrome c1 is rather more reduced than cytochrome c during the oxidation of succinate and of ascorbate + N,N,N',N'-tetramethyl-p-phenylene diamine in both mitochondria and submitochondrial particles; a near equilibrium condition exists between cytochromes c1 and c in the aerobic steady state, with a rate constant for the c1 leads to c reduction step greater than 10(3) s-1. 4. The greater apparent response of the c/aa3 electron transfer step to salts, the hyperbolic inhibition of succinate oxidation by azide and cyanide, and the kinetic behaviour of the succinate-cytochrome c reductase system, are all explicable in terms of a near-equilibrium condition prevailing at the c1/c step. Endogenous cytochrome c of mitochondria and submitochondrial particles is apparently largely bound to cytochrome aa3 units in situ. Cytochrome c1 can either reduce the cytochrome c-cytochrome aa3 complex directly, or requires only a small extra amount of cytochrome c to carry the full electron transfer flux.
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1. The EPR signal in the g = 2 region of the reduced QH2: cytochrome c oxidoreductase as present in submitochondrial particles and the isolated enzyme is an overlap of two signals in a 1 : 1 weighted ratio. Both signals are due to [2Fe-2S]+1 centers. 2. From the signal intensity it is computed that the concentration of each Fe-S center is half that of cytochrome c1. 3. The line shape of one of the Fe-S centers, defined as center 1, is reversibly dependent on the redox state of the b-c1 complex. The change of the line shape cannot be correlated with changes of the redox state of any of the cytochromes in QH2: cytochrome c oxidoreductase. 4. Lie the optical spectrum, the EPR spectrum of the cytochromes is composed of the absorption of at least three different b cytochromes and cytochrome c1. 5. The molar ratio of the prosthetic groups was found to be c1 : b-562 : b-566 : b-558 : center 1 : center 2 = 2 : 2 : 1 : 1 : 1 : 1. The consequences of this stoichiometry are discussed in relation to the basic enzymic unit of QH2 : cytochrome c oxidoreductase.
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The steady-state reduction of exogenous ubiquinone-2 by duroquinol as catalysed by the ubiquinol: cytochrome c oxidoreductase was studied in bovine heart mitoplasts. The reduction of ubiquinone-2 by duroquinol proceeds both in the absence of inhibitors of the enzyme, in the presence of outside inhibitors, e.g., myxothiazol, and in the presence of inside inhibitors, e.g., antimycin, but not in the presence of both inside and outside inhibitors. It is concluded that both the Qin-binding domain and the Qout-binding domain may independently catalyse this reaction. The rate of the reduction of ubiquinone-2 by duroquinol via the Qin-binding domain is dependent on the type of outside inhibitor used. The maximal rate obtained for the reduction of ubiquinone-2 by DQH2 via the Qout-binding domain, measured in the presence of antimycin, is similar to that catalysed by the Qin-binding domain of the non-inhibited enzyme and depends on the redox state of the high-potential electron carriers of the respiratory chain. The reduction of ubiquinone-2 by DQH2 via the Qin-binding domain can be described by a mechanism in which duroquinol reduces the enzyme, upon which the reduced enzyme is rapidly oxidized by ubiquinone-2 yielding ubiquinol-2. By determination of the initial rate under various conditions and simulation of the time course of reduction of ubiquinone-2 using the integrated form of the steady-state rate equation the values of the various kinetic constants were calculated. During the course of reduction of ubiquinone-2 by duroquinol in the presence of outside inhibitors only cytochrome b-562 becomes reduced. At all stages during the reaction, cytochrome b-562 is in equilibrium with the redox potential of the ubiquinone-2/ubiquinol-2 couple but not with that of the duroquinone/duroquinol couple. At low pH values, cytochrome b-562 is reduced in a single phase; at high pH separate reduction phases are observed. In the absence of inhibitors three reduction phases of cytochrome b-562 are discernible at low pH values and two at high pH values. In the presence of antimyin cytochrome b becomes reduced in two phases. Cytochrome b-562 is reduced in the first phase and cytochrome b-566 in the second phase after substantial reduction of ubiquinone-2 to ubiquinol-2 has occurred. In ubiquinone-10 depleted preparations, titration of cytochrome b-562, in the presence of myxothiazol, with the duroquinone/duroquinol redox couple yields a value of napp = 2, both at low and high pH.(ABSTRACT TRUNCATED AT 400 WORDS)
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Some spectral and kinetic properties of the reconstituted succinate oxidase system of Keilin and King have been studied.1.With the reconstituted enzyme system the total rate of succinate oxidation was not affected by dilution.2.The distinct absorption peaks that appeared at 603, 562, 552 and 442 mμ, are essentially the same as those observed for the untreated heart-muscle preparation except for a slight shift of the α-peak of cytochrome a from 604 to 603 mμ.3.A partial inactivation and/or destruction of the cytochromes may have occurred during the alkali treatment of heart-muscle preparation as indicated by the decrease in the extent of enzymic reduction by succinate as well as their low turnover number.4.The velocity constants for the sequential reactions of succinate oxidase have been calculated. The magnitudes of these constants are quite similar to those obtained for the untreated heart-muscle preparation.5.Kinetic evidence indicates that the soluble fraction derived from the alkali treatment of the heart-muscle preparation has a competitive inhibitory effect in relation to the active soluble succinate dehydrogenase (succinate:(acceptor) oxido-reductase, EC 1.3.99.1) on the reduction of cytochrome b by succinate.6.The molecular relationship between succinate dehydrogenase and cytochrome b as well as the participation of cross-linked respiratory chain assemblies during succinate oxidation are discussed.
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The pre-steady-state kinetics of the reduction of the prosthetic groups of QH2:cytochrome c oxidoreductase in bovine heart submitochondrial particles were studied in relation to the kinetics of the Q-10 reduction, using duroquinol as substrate. The prosthetic groups, including semiquinone, were measured with EPR and low-temperature-diffuse reflectance spectroscopy, the samples being prepared with the rapid-freeze quench technique. For the determination of the redox state of ubiquinone in the pre-steady state the rapid chemical quench technique was used as an extension of the rapid-freeze quench technique, and Q-10 and QH2-10 were measured with reversed-phase HPLC after extraction with petroleum ether. Ubiquinone was reduced biphasically, 8% of total Q-10 (equal to 1 mol Q-10/mol cytochrome c1), being reduced within 5 ms, and the rest, the Q-pool, at a much lower rate. The initial rapid reduction of this special Q-10 was accompanied by rapid formation of Qi and rapid reduction of a large part of the cytochrome b-562. Both semiquinone formation and reduction of b-562 showed transient kinetics due to a contribution of the reaction pathway via centre o when the iron-sulphur cluster and cytochrome c1 were oxidised. The majority of the special quinol was located at centre i, probably bound, but also at centre o some bound quinol was formed. This was visible when antimycin was present, the antimycin-insensitive bound quinol being totally sensitive to myxothiazol. Myxothiazol alone accelerated the reduction of the Q-pool via centre i, but also the equilibration of cytochrome b-562 with the Q-pool. Antimycin drastically lowered the rate of reduction of the Q-pool and additionally seemed to block the rapid electron transfer from part of the Rieske iron-sulphur cluster to cytochrome c1. It is concluded that, during the pre-steady-state, cytochrome b-562 is not in equilibrium with the Q-pool and that the rate of equilibration is probably determined by the rate of dissociation of the special bound quinol from centre i.
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Membrane vesicles of physiological as well as inverted orientation can be isolated from mitochondria. The presence of these vesicles in a membrane can be determined and quantitated by determining the differences between the two vesicle types in terms of rates of NADH oxidation, rates of oxidation of tricarboxylate cycle intermediates, rates of ATP hydrolysis and sensitivity to inhibitors, stimulation of respiration by exogenous cytochrome c, inhibition of respiration by polycationic proteins, and visualization of the ATPase by electron microscopy. Procedures to isolate the two membrane types and characteristics of homogeneously oriented preparations are described. Differences in data obtained with homogeneous vesicle preparations and with vesicles of mixed orientation are illustrated. Nonhomogeneously oriented preparations can be enriched in the desired vesicular type by the use of immunoprecipitation, affinity chromatography, and differential centrifugation. The concept of a hybrid vesicle containing oppositely oriented regions is not supported by experimental data.
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Studies have been made of the influence of salts and polyions on several activities of water-washed mitochondria and heart-muscle particles: cytochrome c oxidase (EC 1.9.3.1), NADH cytochrome c reductase and NADH oxidase, Polycations inhibit all enzyme activities involving reaction with soluble cytochrome c to about the same extent. The NADH oxidase activity of heart-muscle particles is not inhibited by amounts of the polycations which completely inhibit the other activities. The cytochrome c oxidase activity of the heart-muscle particles is very sensitive to the concentration of salts in the reaction medium, while the NADH cytochrome c reductase activity is relatively insensitive. The inhibition of the cytochrome c oxidase activity of water-washed mitochondria, but not of heart-muscle particles, increases on preincubation of the preparations with polycations and increases still more rapidly during turnover of the oxidase. Large quantities of cytochrome c are adsorbed by both mitochondria and heart-muscle particles at low ionic strength. There appears to be a rapid exchange between the adsorbed cytochrome c and the cytochrome c in solution. The data emphasize the differences in structure of the respiratory chain system of swollen mitochondria and small membrane fragments and show several aspects of the reaction of the endogenous cytochrome c of the mitochondrial system with neighboring members of the electron-transport chain.
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The rapid reduction and oxidation of horse heart cytochrome c has been studied in the pH range 7 to 12 by stopped flow spectrophotometry. Above pH 8 both the oxidation of ferrocytochrome c by ferricyanide, and the reduction of ferricytochrome c by dithionite, show biphasic kinetics in which electron transfer occurs rapidly in the first phase with a rate dependent on oxidant or reductant concentration. The rate of the second phase is independent of the reductant or oxidant concentration, and the amplitude of the absorbance changes is pH-dependent with an apparent pK near 9.3. Reduction of ferricytochrome c by dithionite at pH 10.7 produces a transient form of ferrocytochrome c characterized by a sharper and more intense Soret peak centered at 418 nm (εm ≥ 173,000), a more intense α-band centered at 550 nm (εm ≥ 31,000) and a reduced β-band near 520 nm (εm ≤ 14,500). During the second phase, this transient form of ferrocytochrome c changes to the classical stable form with a rate constant of 2 to 8 s⁻¹, depending upon the pH. Rapid oxidation of ferrocytochrome c by ferricyanide at pH 10.5 produces a transient species of ferricytochrome c with a 695-nm band absorption and an EPR spectrum characteristic of ferricytochrome c at neutral pH. With time, both the EPR spectrum and the 695-nm band absorption change to the forms normally observed at pH 10.5, and the rate constant of this change is 0.8 s⁻¹. A model is proposed which features a change in the sixth ligand of cytochrome c as being responsible for the changes observed during the second phase.
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1. Treatment of phosphorylating ATP-Mg particles with antimycin in ethanolic solution results in a shift of the signal of low-spin ferric haem of cytochrome b-562 from g = 3.44 to 3.48. This shift is not caused by the ethanol.2. The addition of ATP to particles treated with ascorbate and antimycin results in a shift of the signal at g = 3.48 back to g = 3.44. This effect of ATP is sensitive to uncoupler. In the absence of antimycin, ATP has no effect on the signal.3. Neither ATP nor antimycin has any effect on the intensity of the signal due to cytochrome b-566.4. Ethanol (5%, v/v) has marked effects on the EPR spectrum of cytochrome c oxidase which have to be taken into account when interpreting the effects of antimycin in ethanolic solution. It causes a shift of the signal of low-spin iron from g = 3.01 to g = 3.05 and a change of height and shape of the signal of high-spin iron at g = 5.85.5. ATP added to particles treated with ascorbate and antimycin causes an increase in intensity of the signal at g = 3.05. This is correlated with an increased oxidation of cytochrome c and c1 and cytochrome c oxidase observed spectroscopically.6. The addition of cyanide to particles or isolated cytochrome c oxidase, in the presence of reducing agent, results in the appearance of a low-spin signal at g = 3.58 in the absence of ethanol and at g = 3.62 in the presence of 5% (v/v) ethanol. This is ascribed to low-spin ferric haem of the cyano compound of cytochrome c oxidase.
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1.1. Fractionation of beef heart submitochondrial membrane fragments derived by sonic disruption was achieved by linear sucrose density gradient centrifugation. Three distinct membrane fractions were obtained: a pellet (P), a middle (X) and an upper (Y) zone.2.2. Electron micrographs revealed that the pellet contained large single pieces and aggregates of membrane fragments. The X zone contained rough membrane fragments with protruding subunits (F1) and the Y zone contained relatively small fragments without subunits.3.3. In comparison with the Y zone, the X zone exhibited higher activities of NADH oxidase and ATPase, higher oligomycin-induced respiratory control and cytochrome content, but lower activities of NADH:cytochrome c reductase (rote-none-sensitive) and cytochrome oxidase with externally-added cytochrome c as the substrate. Lubrol greatly enhanced the cytochrome oxidase activity in the X zone but not in the Y zone. NADH oxidase was greatly stimulated by externally added cytochrome c in the Y zone but not in the X zone. In general, the pellet exhibited varying activities falling between those of the X zone and the Y zone.4.4. The relationship between these membrane fractions and the conventional EDTA particle preparation is discussed.
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Cytochromec added during the formation of lecithin-cardiolipin liquid crystals in 0.015M KCl is readily bound. After successive washings with 0.15M KCl, only about 50% of this bound cytochromec is removed. The remaining cytochromec is resistant to further salt extraction, and the amount of this cytochromec that is bound varies with the concentration of added cytochromec to a maximum binding ratio of 1∶70, mole ratio cytochromec to phospholipid. This binding appears to be electrostatic; it is competitively inhibited by increasing the initial molarity of KCl from 0.015 to 0.10M. Binding of cytochromec is insignificant in the absence of cardiolipin, and is affected by varying the pH. Electron microscope studies of osmium tetroxide-stained thin sections show that the liquid crystals consist of vesicles, each of which contains a large number of concentric, alternating light and dense lines. The dense lines have been identified by other workers with the polar head groups of the phospholipids on the surface of a bilayer, and the light area represents the hydrophobic interior. The addition of cytochromec causes an average decrease in the number of lines per vesicle. It increases the center-to-center distance between two neighboring light or dense lines and the width of the dense lines. On the basis of this evidence and electrostatic binding, it is concluded that cytochromec is binding on the polar surfaces of the phospholipid bilayers comprising the liquid crystalline vesicles.
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The extent of inhibition of cytochrome oxidase activity by cyanide depends upon the method of assay. It has been confirmed that this inhibition is “readily reversible” with the use of the standard manometric technique. However, in the spectrophotometric assay only about 10% of the control activity is observed after the cyanide is removed by physical methods. The polarographic assay yields intermediate results. On the other hand, the polarographic assay shows a complete reversal of inhibition when the cyanide-treated heart muscle preparation is first incubated under reducing conditions. The tetrachlorohydroquinone oxidase activity of the heart muscle preparation is also decreased by cyanide treatment. The spectra of the purified cytochrome oxidase treated with cyanide are similar to those of cytochrome a. It is likely that there are two sites for the interaction of cytochrome oxidase and cyanide: the heme a iron and a second noncovalent locus. The nature of this second site s discussed.
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In the 1950s, the studies of proteins through their synthesis captured the attention of a number of biochemists. Among teams that set out to chemically synthesize the protein insulin, a large team in the People’s Republic of China achieved success in 1966, months before the Cultural Revolution. By focusing on the ideological refashioning, material arrangement, and organizational style of the project, this paper addresses the political and material dimensions of the project, especially how it was reconstructed as an engineering project in-between biology and chemistry for the young republic. This case was different from the design rationales demonstrated in both American and German cases, in which insulin synthesis was viewed as either a challenging problem for biochemistry or primary research toward making synthetic fibers. The process reveals a fluid topography of the material, social, and political space that a group of biochemists could work with in socialist China.
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Mitochondria isolated from the thymus of rats or rabbits 4 hours after 800 rads of ionizing radiation show uncoupling of their oxidative phosphorylation. There are no significant differences in the contents of phospholipids, neutral lipids, or total fatty acids between normal and uncoupled mitochondria. There was no evidence of the formation of any lipid uncoupling factor in thymus mitochondria from irradiated animals.Heart or liver mitochondria isolated from animals irradiated up to 12 hours previously showed no uncoupling of oxidative phosphorylation although preparations in various media could be made with reduced cytochrome c contents. No effect of irradiation upon endogenous cytochrome c or its binding in these mitochondria could be detected.Thymus mitochondria would appear to contain very low quantities of cytochrome c (less than 0.2 μg per mg protein), yet still be tightly coupled. It is suggested that this low level may indicate a critical condition at which radiation damage could become manifest.
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In der tierischen Zelle laufen Hunderte von chemischen Reaktionen ab. Fast alle diese Vorgänge werden durch Fermente (Synonyme: Enzym, diastase) beschleunigt oder überhaupt erst ermöglicht. Da die einzelnen chemischen Abläufe untereinander zu einem reichen Reaktionsnetzwerk verknüpft sind, so ergibt sich, daß selbst die wenigen chemischen Prozesse, die nicht fermentativ gesteuert werden, mittelbar doch durch Enzyme beeinflußt werden. Es ist somit nicht denkbar, ein umfassendes Verständnis für die Lebensvorgänge ohne Berücksichtigung der Fermente zu gewinnen. Selbst der Stoffaustausch zwischen der Zelle und ihrer Umgebung ist mit Fermentreaktionen verbunden. Dasselbe gilt auch für den Aufbau und für die Erhaltung der Zellstrukturen. Ohne ständige fermentative Resynthese würden die Bauelemente der Zelle, die sich in einem unstabilen Zustand befinden, bald zusammenbrechen. Ein tieferes Erfassen der Wirkungsweise vieler Hormone, Vitamine, Gifte, Pharmaka und Chemotherapeutica ist ohne das Studium des Einflusses dieser Stoffe auf Fermente ebenfalls kaum denkbar. Diese Aussage gilt selbst dann, wenn der primäre Angriffspunkt einiger Wirkstoffe nicht ein Enzym sein sollte.
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It is likely that, within the intact cell, the freedom of movement of all enzymes is restricted. To that extent they could all be considered structurally-bound. However, from the purely experimental point of view, it is a striking fact that when a cell is ruptured, a number of enzymes go into solution, while others remain firmly bound to more or less large particles, which can be sedimented in ordinary centrifuges. It is these enzymes which we think of when we refer to structurally-bound enzymes.
1. Mitochondria isolated from rat liver were disrupted with 0.3 per cent deoxycholate and a number of subfractions were isolated from this preparation by differential centrifugation. 2. The protein N, RNA and phospholipide content, as well as the succinoxidase, cytochrome c oxidase, adenylate kinase, and DPNH-cytochrome c reductase of these fractions were determined. 3. Two of these subfractions, found to consist of mitochondrial membranes (2), contained ∼ 12 per cent of the protein N and ∼ 35 per cent of the phospholipide of the whole mitochondria and accounted for ∼ 70 per cent of the succinoxidase and cytochrome c oxidase activity of the original mitochondrial preparation. There was no discernible adenylate kinase, DPNH-cytochrome c reductase, or phosphorylating activities in these fractions, nor could they oxidize other substrates of the Krebs's cycle. 4. The most active fraction (60 minutes at 105,000 g pellet) had a higher phospholipide/protein value than the whole mitochondria and showed a seven-to elevenfold concentration of succinoxidase and cytochrome c oxidase activities. 5. Evidence has been given to indicate that the various components of the succinoxidase complex are present in this membrane fraction in the same relative proportions as in the whole mitochondria. 6. The implications of these findings are discussed.
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This and the companion article are aimed at surveying the methods used for the study of membrane asymmetry. The techniques employed for the assessment of the asymmetric distribution and orientation of membrane proteins are reviewed in this article, whereas those pertaining to the unequal distribution of lipids are detailed in the companion paper. The use of immunological techniques and lectins, functions of protein and their perturbations, chemical reagents, enzymatic isotopic labeling and enzymatic cleavage of membrane proteins and physical techniques are discussed and illustrated using recent examples of their application. Whenever appropriate, problems involving crypticity and non-availability or non-reactivity of functional sites, relevant chemical functions or protein fragments to appropriate ligands, reagents or modifying enzymes are envisaged and possible modification of the exposure of proteins during preparation of ghosts and other drawbacks are discussed, the use of different techniques and control experiments in conjunction is recommended for a more realistic assessment of the distribution and orientation of proteins.
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Cytochromec can be bound to mixed cardiolipin-lecithin liquid crystals so that it cannot be removed by repeated washings with solutions of high ionic strength. The oxidized and reduced spectra of this cytochromec show no detectable differences from those for soluble cytochromec. Unlike soluble cytochromec, however, some 90% of the bound cytochromec is not reduced by ascorbate, and it is only slowly reduced by dithionite. The addition of redox dyes causes complete and immediate reduction in the presence of ascorbate or dithionite. It is suggested that this is because the dyes possess some degree of lipid solubility and are able to penetrate the phospholipid membrane barriers separating cytochromec from the bulk solution. The addition of detergents, such as Triton X-100, also promotes reduction of the bound cytochromec by ascorbate. A small change in the standard potential from, 273 mV for soluble cytochromec to 225 mV for the bound cytochromec was found. The bound cytochromec reacts readily with potassium cyanide to form the normal cyanide-ferricytochromec complex, differing in the rate of formation from soluble cytochromec only at alkaline pH values. The relationship of these findings to work on the membrane-bound cytochromec in mitochondria and submitochondrial particles is discussed.
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Introduction Development Isolation Properties Primary, Secondary, and Tertiary Structures Electron Transfer Cytochrome c1 Complexes One-band Cytochrome c1 Concluding Remarks
Article
The principal intracytoplasmic form of muscle mitochondria is rod shaped and irregular. The mitochondria of muscle are easily stained by the variety of techniques peculiar to mitochondrial staining, such as the procedures of Regaud, Altman, and Benda, and by Baker's method. The absence of structural membranes would bring the matrix of the mitochondrion into contiguity with the sarcoplasm and the adjacent myofibrillae. In transverse sections of skeletal muscle an interpenetration of myofibrillary substance and mitochondrial matrix is demonstrated. In addition to the approximation by contact, the mitochondria are linked to both the myofibrillae and the sarcosomes by delicate reticular strands. The ability of the muscle cyclophorase from both cardiac and skeletal muscles to oxidize all the citric acid substrates catalytically is not precisely paralleled by the mitochondria in pure suspension. There is sluggish citrate oxidation in particular in the cardiac mitochondria and some lag in skeletal muscle. This has been associated with diminution in the apparent isocitric acid dehydrogenase activity in mitochondria, because most of the activity of this enzyme observed in homogenates is recovered in the supernatant fraction.
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Equilibrium and kinetic studies of the isomerization of horse heart ferricytochrome c at alkaline pH have been carried out using difference spectroscopy and stopped flow techniques. The minimal reaction scheme is: [see PDF for equation] where CH and C are species of ferricytochrome c which exhibit a 695 nm absorbance band, and NC (no color) is a form without this band; the oxidation-reduction potentials of CH and NC differ significantly. The values of the constants in this scheme were determined at 25° except for the oxidation and reduction rate constants, which were determined at 22°. The pH dependence of the observed rate constant for the conformational changes was measured as the change in the 695 nm band absorbance and as the release and uptake of hydrogen ions. From this it was calculated that the ionizing group in the scheme above has an apparent pK of 11.0 ± 0.1 at 25°. The conformation equilibrium constant, Kc, defined as κf/κb, was found to be 125 ± 36 at 25°. An over-all pK of 9.0 was calculated from the product of the equilibrium constants, in agreement with the over-all pK of 9.0 determined from the pH dependence of the 695 nm band at equilibrium. Spectrophotometric measurements at 243 nm indicate that tyrosine is not the ionizing group with an apparent pK of 11. It is suggested that the difference in oxidation-reduction potential and the absorbance band at 695 nm arises by substituting the sulfur of Met-80 as one of the ligands of the iron porphyrin in CH by the ε-amino group of Lys-79 in NC. This suggestion, consistent with structural information, is accessible to experimental verification.
Article
Particulate preparations from pigeon breast muscle have been found to require the addition of a factor for maximum activity in the oxidation of succinate. This factor is found in the supernate from the enzyme preparation and has been identified after electrophoretic separation as cytochrome c. This method of identification is particularly useful when other coloured compounds are present in the supernate. Unlike cytochrome c addition of excess of the supernate S1 inhibits the succinoxidase system.RésuméDes préparations de particules de muscle pectoral de pigeon nécessitent l'addition d'un certain facteur pour que l'oxydation du succinate y ait lieu avec l'activité maximum. Ce facteur se trouve dans le surnageant des préparations enzymatiques et a été identifié, après séparation par électrophorèse, avec le cytochrome c. Cette méthode d'identification est particulièrement utile lorsque d'autres produits colorés sont présents dans le surnageant. A la différence du cytochrome c, l'addition d'un excès du surnageant S1 inhibe le système succinoxydasique.ZusammenfassungEs wurde beobachtet, dass partikeln aus Taubenbrustmuskeln die Hinzufügung eines Faktors für eine Höchstaktivität in der Oxydation von Succinat benötigen. Dieser Faktor wird in der überstehenden Flüssigkeit des Enzympräparates gefunden und konnte, nach elektrophoretischer Separation, als Cytochrom c identifiziert werden. Diese Identifikationsmethode ist besonders nützlich, wenn andere farbige Körper in der überstehenden Flüssigkeit zugegen sind. Im Gegensatz zu Cytochrom c hemmt die Hinzufügung eines Überschusses an überstehender Flüssigkeit S1 das Succinoxydasesystem.
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IntroductionConstitution and Preparations of the Respiratory ChainGeneral Concepts of Organization and ReconstitutionReconstitution Employing Lipid-Free, Soluble Components and ParticlesReconstitution Employing Lipid-Containing ParticlesComparison of Reconstitution Work Done in Various LaboratoriesAn Inquiry into the Linkages between the Respiratory ComponentsConcluding Remarks
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A cytochrome c derivative from which iron is removed has been prepared and characterized. Several lines of evidente indicate that native and porphyrin cytochrome c have similar conformations: they have similar elution characteristics an Sephadex gel chromatography; in both proteins the tryptophan fluorescence is quenched and the pK values of protonation of the porphyrin are identical. Porphyrin cytochrome c does not substitute for native cytochrome c in Bither the oxidase reaction or in restoring electron transport in cytochrome-c-depleted mitochondria. It does however competitively inhibit native cytochrome c in these reactions, the Ki for inhibition being larger than the Km for reaction. The absorption and emission spectra, and the polarized excitation spectrum of the porphyrin cytochrome c are characteristic of free base porphyrin. The absente of fluorescence quenching of porphyrin cytochrome c when the protein is bound to cytochrome oxidase suggests that heure to heure distance between these proteins is larger than 0.5 to 0.9 nm depending upon orientation. Binding of the porphyrin cytochrome c to phospholipids or to mitochondria increases the fluorescence polarization of a positively polarized absorption band, which indicates that the bound form of the protein does not rotate freely within the time scale of relaxation from the excited state
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This chapter discusses a new technique of differential centrifugation in the study of cellular organization and the theoretical basis of the technique as well as the various factors of a practical nature. The limitations of differential centrifugation become particularly severe when the technique is applied to the study of tissue enzymes. The observed partitions provide only the roughest sort of information concerning the true intracellular distribution of enzymes. They can only be considered as clues, which have to be followed by many additional experiments to arrive at their real significance. A priori assumption that specific enzymes are entirely concentrated in a given cellular site has proved extremely profitable in guiding these additional experiments, even in those cases in which it is found not to hold true. The use of enzyme determinations to ascertain the composition of isolated fractions is also of great interest and deserves more frequent application. The main object of cellular physiology is to ascertain which of the many processes discovered by biochemists actually do take place within the cell.
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1. Based on the assumptions that the quenching of the fluorescence of antimycin on binding to the bc1 segment of the respiratory chain (Complex III) is caused by energy transfer from antimycin to the cytochrome b haem, and the intrinsic fluorescence of antimycin bound to the complex is the same as that in ethanolic solution the distance between the fluorescent group of antimycin and the b haem was found to be 1.9 nm in oxidized Complex III and 2.4 nm in the reduced Complex. If the intrinsic enhancement of the fluorescence on binding to the Complex is the same as that on binding to serum albumin (about 9-fold), these distances become 1.35 nm and 1.7 nm, respectively.