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2.
1. In mitochondrial particles antimycin binds to two separate specific sites with dissociation constants KD1 less than 4 - 10(-13) M and KD2 = 3 - 10(-9) M, respectively. 2. The concentrations of the two antimycin binding sites are about equal. The absolute concentration for each binding site is about 100 - 150 pmol per mg of mitochondrial protein. 3. Antimycin bound to the stronger site mainly inhibits NADH-and succinate oxidase. Binding of antimycin to the weaker binding site inhibits the electron flux to exogenously added cytochrome c after blocking cytochrome oxidase by KCN. 4. Under certain conditions cytochrome b and c1 are dispensible components for antimycin-sensitive electron transport. 5. A model of the respiratory chain in yeast is proposed which accounts for the results reported here and previously. (Lang, B., Burger, G., and Bandlow, W. (1974) Biochim. Biophys. Acta 368, 71-85). 相似文献
4.
1. Beef heart mitochondria have a cytochrome c1 : c : aa3 ratio of 0.65 : 1.0 : 1.0 as isolated; Keilin-Hartree submitochondrial particles have 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° – 30°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 → c reduction step greater than 10 3 s ?1.4. The greater apparent response of the 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 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. 相似文献
5.
1. Formate inhibits cytochrome oxidase activity both in intact mitochondria and submitochondrial particles, and in isolated cytochrome . The inhibition increases with decreasing pH, indicating that HCOOH may be the inhibitory species.2. Formate induces a blue shift in the absorption spectrum of oxidized cytochrome ) and in the half-reduced species ( ). Comparison with cyanide-induced spectral shifts, towards the red, indicates that formate and cyanide have opposite effects on the spectrum, both in the fully oxidized and the half-reduced states. The formate spectra provide a new method of obtaining the difference spectrum of minus , free of the difficulties with cyanide (which induces marked high → low spin spectral shifts in cytochrome ) and azide (which induces peak shifts of cytochrome towards the blue in both α- and Soret regions).3. The rate of formate dissociation from cytochrome is faster than its rate of dissociation from , especially in the presence of cytochrome . The for formate inhibition of respiration is a function of the reduction state of the system, varying from 30 mM (100% reduction) to 1 mM (100% oxidation) at pH 7.4, 30 °C.4. Succinate-cytochrome reductase activity is also inhibited by formate, in a reaction competitive with succinate and dependent on [formate] 2.5. Formate inhibition of ascorbate plus oxidation by intact rat liver mitochondria is partially released by uncoupler addition. Formate is permeable through the inner mitochondrial membrane and no differences in ‘on’ or ‘off’ inhibition rates were observed when intact mitochondria were compared with submitochondrial particles.6. NADH-cytochrome reductase activity is unaffected by formate in submitochondrial particles, but mitochondrial oxidation of glutamate plus malate is subject both to terminal inhibition at the cytochrome level and to a slow extra inhibition by formate following uncoupler addition, indicating a third site of formate action in the intact mitochondrion. 相似文献
7.
Several components of the respiratory chain of the eubacterium Thermus thermophilus have previously been characterized to various extent, while no conclusive evidence for a cytochrome bc1 complex has been obtained. Here, we show that four consecutive genes encoding cytochrome bc1 subunits are organized in an operon-like structure termed fbcCXFB. The four gene products are identified as genuine subunits of a cytochrome bc1 complex isolated from membranes of T. thermophilus. While both the cytochrome b and the FeS subunit show typical features of canonical subunits of this respiratory complex, a further membrane-integral component (FbcX) of so far unknown function copurifies as a subunit of this complex. The cytochrome c1 carries an extensive N-terminal hydrophilic domain, followed by a hydrophobic, presumably membrane-embedded helical region and a typical heme c binding domain. This latter sequence has been expressed in Escherichia coli, and in vitro shown to be a kinetically competent electron donor to cytochrome c552, mediating electron transfer to the ba3 oxidase. Identification of this cytochrome bc1 complex bridges the gap between the previously reported NADH oxidation activities and terminal oxidases, thus, defining all components of a minimal, mitochondrial-type electron transfer chain in this evolutionary ancient thermophile. 相似文献
9.
The steady-state kinetics of purified yeast cytochrome c oxidase were investigated at low ionic strength where the electrostatic interaction with cytochrome c is maximized. In 10 mM cacodylate/Tris (pH 6.5) the oxidation kinetics of yeast iso-1-cytochrome c were sigmoidal with a Hill coefficient of 2.35, suggesting cooperative binding. The half-saturation point was 1.14 μM. Horse cytochrome c exhibited Michaelis-Menten kinetics with a higher affinity ( Km = 0.35 μM) and a 100% higher maximal velocity.In 67 mM phosphate the Hill coefficient for yeast cytochrome c decreased to 1.42, and the species differences in Hill coefficients were lessened. Under the latter conditions, a yeast enzyme preparation partially depleted of phospholipids was activated on addition of diphosphatidylglycerol liposomes. When the enzyme was incorporated into sonicated yeast promitochondrial particles the apparent Km for horse cytochrome c was considerably lower than the value for the isolated enzyme.ATP was found to inhibit both the isolated oxidase and the membrane-bound enzyme. With the isolated enzyme in 10 mM cacodylate/Tris, 3 mM ATP increased the half-saturation point with yeast cytochrome c 3-fold, without altering the maximal velocity or the Hill coefficient. 67 mM phosphate abolished the inhibition of the isolated oxidase by ATP.The increase in affinity for cytochrome c produced by binding the oxidase to the membrane was not observed in the presence of 3 mM ATP, with the result that the membrane-bound enzyme was more sensitive to inhibition by ATP. ADP was a less effective inhibitor than ATP, and did not prevent the inhibition by ATP.It is proposed that non-specific electrostatic binding of cytochrome c to phospholipid membranes, followed by rapid lateral diffusion, is responsible for the dependence of the affinity on the amount and nature of the phospholipids and on the ionic strength.ATP may interfere with the membrane-facilitated binding of cytochrome c by a specific electrostatic interaction with the membrane or by binding to cytochrome c. 相似文献
10.
We have compared the efficacy of inhibition of the cytochrome bc1 complexes from yeast and bovine heart mitochondria and Paracoccus denitrificans by antimycin, ilicicolin H, and funiculosin, three inhibitors that act at the quinone reduction site at center N of the enzyme. Although the three inhibitors have some structural features in common, they differ significantly in their patterns of inhibition. Also, while the overall folding pattern of cytochrome b around center N is similar in the enzymes from the three species, amino acid sequence differences create sufficient structural differences so that there are striking differences in the inhibitors binding to the three enzymes. Antimycin is the most tightly bound of the three inhibitors, and binds stoichiometrically to the isolated enzymes from all three species under the cytochrome c reductase assay conditions. Ilicicolin H also binds stoichiometrically to the yeast enzyme, but binds approximately 2 orders of magnitude less tightly to the bovine enzyme and is essentially non-inhibitory to the Paracoccus enzyme. Funiculosin on the other hand inhibits the yeast and bovine enzymes similarly, with IC 50 ∼ 10 nM, while the IC 50 for the Paracoccus enzyme is more than 10-fold higher. Similar differences in inhibitor efficacy were noted in bc1 complexes from yeast mutants with single amino acid substitutions at the center N site, although the binding affinity of quinone and quinol substrates were not perturbed to a degree that impaired catalytic function in the variant enzymes. These results reveal a high degree of specificity in the determinants of ligand-binding at center N, accompanied by sufficient structural plasticity for substrate binding as to not compromise center N function. The results also demonstrate that, in principle, it should be possible to design novel inhibitors targeted toward center N of the bc1 complex with appropriate species selectivity to allow their use as drugs against pathogenic fungi and parasites. 相似文献
12.
1. Extraction by ether removes only about one-half of antimycin added to sub-mitochondrial particles, independently of the amount of antimycin added up to that necessary for 100% inhibition. 2. The amount of antimycin extractable with ether remains the same even when the antimycin is redistributed between an antimycin-inhibited preparation and an untreated. The antimycin remaining after ether extraction is redistributed between ether-accessible and ether-inaccessible sites when the preparation is incubated on its own or with an untreated preparation. 3. Low concentrations of cholate increase the extractability of the antimycin by ether. 4. Complex III binds antimycin more firmly than sub-mitochondrial particles. However, antimycin is readily extracted by ether, leading to restoration of enzymic activity and cleavage of the complex by bile salts. 5. The results are consistent with an explanation of the sigmoidal inhibition curve with antimycin and preparations of the intact respiratory chain in terms of an allosteric model. 相似文献
14.
1. Purified mitochondria have been prepared from wild type Paramecium tetraurelia and from the mutant Cl 1 which lacks cytochrome aa3. Both mitochondrial preparations are characterized by cyanide insensitivity. Their spectral properties and their redox potentials have been studied.2. Difference spectra (dithionite reduced minus oxidized) of mitochondria from wild type P. tetraurelia at 77 K revealed the α peaks of b-type cytochrome(s) at 553 and 557 nm, of c-type cytochrome at 549 nm and a-type cytochrome at 608 nm. Two α peaks at 549 and 545 nm could be distinguished in the isolated cytochrome c at 77 K. After cytochrome c extraction from wild type mitochondria, a new peak at 551 nm was unmasked, probably belonging to cytochrome c1. The a-type cytochrome was characterized by a split Soret band with maxima at 441 and 450 nm. The mitochondria of the mutant Cl 1 in exponential phase of growth differed from the wild type mitochondria in that cytochrome aa3 was absent while twice the quantity of cytochrome b was present. In stationary phase, mitochondria of the mutant were characterized by a new absorption peak at 590 nm.3. Cytochrome aa3 was present at a concentration of 0.3 nmol/mg protein in wild type mitochondria and ubiquinone at a concentration of 8 nmol/mg protein both in mitochondria of the wild type and the mutant Cl 1. Cytochrome aa3 was more susceptible to heat than cytochromes b and c, c1.4. CO difference spectra at 77 K revealed two different Co-cytochrome complexes. The first, found only in wild type mitochondria, was a typical CO-cytochrome a3 complex characterized by peaks at 596 and 435 nm and troughs at 613 and 450 nm. The second, found both in mitochondria of the wild type and the mutant, was a CO-cytochrome b complex with peaks at 567, 539 and 420 nm and a trough at 558-549 nm. Both complexes are photo-dissociable.5. Spectral evidence was obtained for interaction of cyanide with the a-type cytochrome (shift of the α peak at 77 K from 608 to 605 nm), but not with the b-type cytochrome.6. The mid-point potentials of the different cytochromes at neutral pH are as follows: cytochrome aa3 235 and 395 mV, cytochrome c, c1 233 mV, cytochromes b 120 mV. 相似文献
15.
1. Pretreatment of sub-mitochondrial particles with cholate results in a change in the curve describing inhibition by antimycin of the succinate-cytochrome c reductase from sigmoidal towards linear. This effect of cholate is reversed by partial removal of the cholate by dialysis, either in the absence or presence of antimycin. 2. Treatment with cholate has the same action on the sigmoidal effect curve of antimycin on the reducibility of cytochrome b. This is also reversed by dialysis. 3. The effect of antimycin on the displacement to the red of the -band of ferrocytochrome b, measured in the presence of succinate, NADH or reduced ubiquinone Q-2, is also described by a sigmoidal curve that is changed to a linear one by addition of cholate. 4. Linear displacement curves are obtained with menaquinol or Na2S2O4. 5. It is proposed that antimycin is an allosteric inhibitor of the respiratory chain. This allosteric effect should be distinguished from the effect of antimycin on the “conformation stability” of Complex III. 相似文献
16.
(1) Cells of ThiobacillusA2 grown chemoautotrophically on thiosulfate or heterotrophically on succinate with oxygen contained b-, c-, o-, a- and a3-type cytochromes. The amount of cytochrome per mg of cell protein was much greater in thiosulfate-grown cells and differences in the relative concentrations of cytochromes were observed for the different growth conditions. (2) The half-reduction potentials at pH 7.0 ( Em,7.0) and spectral maxima of c-, b-, a- and a3-type cytochromes were similar in cells grown aerobically with thiosulfate or with succinate as the growth substrate. (3) The half-reduction potential of the ‘invisible’, or high-potential copper, as determined from the potentiometric behavior of the carbon monoxide-reduced cytochrome a3 complex at pH 8.0, was 365 mV. (4) Reducing equivalents from thiosulfate appear to enter the respiratory chain at the cytochrome c level; however, studies in cell-free extracts were limited due to a loss in respiratory activity with thiosulfate as a substrate upon cell disruption. 相似文献
17.
The binding of antimycin was studied in several mutant strains of yeast that have specific defects in cytochrome b. The strains have mutations in a part of the mitochondrial DNA that contains the structural gene for the apoprotein of cytochome b. Two of the mutants lack this protein and have no spectral cytochrome b. These mutants also lack the strong antimycin-binding site that is present in wild-type yeast mitochondria in the ratio of one site per two cytochrome b molecules. A third mutant which contains normal levels of spectral cytochrome b, but shows an altered absorption maximum for cytochrome b at 77 °K, was found to bind normal amounts of antimycin. However, the fluorescence of antimycin bound to mitochondria of this strain was found to be less efficiently quenched than in the case of the wild-type strain. In another mutant which contains only 20% of the normal spectral level of cytochrome b, the number of antimycin-binding sites was proportionately less. In an antimycin-resistant mutant, the binding of antimycin was too weak to be detected. The simultaneous modification of the structure of cytochrome b and the alteration of the antimycin-binding site in these mutants suggests that the antimycin-binding site is located on the apoprotein of cytochrome b. 相似文献
18.
Electronic connection between Q o and Q i quinone catalytic sites of dimeric cytochrome bc1 is a central feature of the energy-conserving Q cycle. While both the intra- and inter-monomer electron transfers were shown to connect the sites in the enzyme, mechanistic and physiological significance of the latter remains unclear. Here, using a series of mutated hybrid cytochrome bc1-like complexes, we show that inter-monomer electron transfer robustly sustains the function of the enzyme in vivo, even when the two subunits in a dimer come from different species. This indicates that minimal requirement for bioenergetic efficiency is to provide a chain of cofactors for uncompromised electron flux between the catalytic sites, while the details of protein scaffold are secondary. 相似文献
19.
We measured production of reactive oxygen species by intact mitochondria from rat skeletal muscle, heart, and liver under various experimental conditions. By using different substrates and inhibitors, we determined the sites of production (which complexes in the electron transport chain produced superoxide). By measuring hydrogen peroxide production in the absence and presence of exogenous superoxide dismutase, we established the topology of superoxide production (on which side of the mitochondrial inner membrane superoxide was produced). Mitochondria did not release measurable amounts of superoxide or hydrogen peroxide when respiring on complex I or complex II substrates. Mitochondria from skeletal muscle or heart generated significant amounts of superoxide from complex I when respiring on palmitoyl carnitine. They produced superoxide at considerable rates in the presence of various inhibitors of the electron transport chain. Complex I (and perhaps the fatty acid oxidation electron transfer flavoprotein and its oxidoreductase) released superoxide on the matrix side of the inner membrane, whereas center o of complex III released superoxide on the cytoplasmic side. These results do not support the idea that mitochondria produce considerable amounts of reactive oxygen species under physiological conditions. Our upper estimate of the proportion of electron flow giving rise to hydrogen peroxide with palmitoyl carnitine as substrate (0.15%) is more than an order of magnitude lower than commonly cited values. We observed no difference in the rate of hydrogen peroxide production between rat and pigeon heart mitochondria respiring on complex I substrates. However, when complex I was fully reduced using rotenone, rat mitochondria released significantly more hydrogen peroxide than pigeon mitochondria. This difference was solely due to an elevated concentration of complex I in rat compared with pigeon heart mitochondria. 相似文献
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