Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.99.3 (diaphorase)
5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chicken heart mitochondrial ubiquinol: cytochrome c reductase has been isolated as a monodisperse form by solubilization with Triton X-100 in low ionic strength and subsequent affinity chromatography. The enzyme complex contains less than 0.15 mumol ubiquinone and 12 mumol phospholipid per mumol enzyme. The spectrum of cytochrome b in the reduced exhibited two maximum alpha-absorption bands at 558 nm and 562 nm; a shoulder at 553 nm for c1. The purified enzyme complex consists of at least 9 subunits with apparent molecular weight of 47 K, 44 K, 36 K, 31 K, 25 K, 17 K, 15 K, 12 K and 9 K. The enzyme complex has been dissociated into subdomains of subunits in the presence of increasing concentration of salt and Triton X-100. Three subunits carrying the redox centers (cytochrome b, cytochrome c1 and Fe-S protein) have been further isolated.
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PMID:Chicken heart ubiquinol: cytochrome c reductase--isolation by affinity chromatography and resolution of enzyme into subunits. 301 91

The reliability of monitoring the redox reactions of cytochrome b using the different wavelengths employed by different authors has been reexamined. It was found that 562-575 nm is suitable in succinate: cytochrome c reductase but not in mitochondria, in which case 562-540 nm is a better pair. Direct optical measurements of the redox reaction kinetics of the mitochondrial Q pool using a commercial dual-wavelength spectrophotometer are possible when succinate is used as the electron donor. Using the correct wavelength pair, and with malonate to slow down the electron input, the reduction course of cytochrome b was still triphasic but a plateau or a turn replaced the oxidation phase previously reported by several authors. At the same time, the reduction course of the Q pool was also triphasic, and in perfect match with that of cytochrome b. Destruction of the Rieske iron-sulfur cluster by British anti-Lewisite (BAL) + O2 treatment or prereduction of the high-potential components made the reduction of both Q and b monophasic. The plot of log (Q/QH2) against log (b3+/b2+) gave a straight line with an n value of 1.7 for cytochrome b at pH 7.4. This n value rose to 2.0 at pH 6.5 and dropped to 1.4 at pH 8.5. On the other hand, the mid-point potential of cytochrome b relative to that of the Q pool remained essentially unchanged between pH 6.5 and 8.4. BAL treatment had a small effect on the midpoint potential of cytochrome b relative to that of the Q pool and had no effect on the n value. Addition of quinone homologues and analogues extended the plateau phase in the reduction of cytochrome b, but exogenous quinones did not equilibrate rapidly with cytochrome b. It was concluded that the appearance of the plateau between the two reduction phases of Q and b is caused by the rapid delivery of electrons to the high-potential components of the respiratory chain as envisaged in the Q cycle; the unexpected n value for cytochrome b suggests a concerted reduction by QH2 of two species of cytochromes b-562.
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PMID:Rapid redox equilibrium between the mitochondrial Q pool and cytochrome b during triphasic reduction of cytochrome b by succinate. 301 94

We have investigated the oxidation of the reduced ubiquinol:cytochrome c reductase (bc1 complex) isolated from beef heart mitochondria. The oxidation of cytochrome c1 by both potassium ferricyanide and cytochrome c in the ascorbate-reduced bc1 complex is not a first-order process. This is taken as evidence that cytochrome c1 is in rapid equilibrium with the Rieske iron-sulphur center. Among the several inhibitors tested, only 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole and stigmatellin are seen to affect this redox equilibrium between the high-potential centers of the beef heart bc1 complex. The oxidation of cytochrome b by cytochrome c in both the succinate-reduced and the fully reduced bc1 complex is blocked by all the inhibitors tested. This inhibition occurs simultaneously with an acceleration in the oxidation of cytochrome c1, even after extraction of the endogenous ubiquinone which is present in the bc1 preparation. Almost complete extraction of ubiquinone from the bc1 complex has no effect upon the rapid phase of cytochrome b oxidation, nor does it alter the inhibition of cytochrome b oxidation by the various inhibitors. The oxidation of cytochrome b by exogenous ubiquinones is stimulated by myxothiazol and partially inhibited by antimycin. However, the addition of both these inhibitors together completely blocks the oxidation of cytochrome b by quinones. In contrast, the simultaneous addition of antimycin and myxothiazol has no such synergistic effect upon the oxidation of cytochrome b by cytochrome c. Our data show that intramolecular electron transfer from cytochrome(s) b to the Rieske iron-sulphur center can take place in the bc1 complex without involvement of endogenous ubiquinone-10. This electron pathway is sensitive to all the inhibitors of the enzyme.
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PMID:On the oxidation pathways of the mitochondrial bc1 complex from beef heart. Effects of various inhibitors. 302 79

A large-scale preparation of the two-subunit protein complex (QPs) that converts succinate dehydrogenase into succinate-ubiquinone reductase from cytochrome b-c1 particles is achieved by a procedure involving Triton X-100 solubilization and calcium phosphate column chromatography at different pH values. The isolated two-subunit QPs contains 25 nmol of cytochrome b560/mg of protein and is able to reconstitute with soluble succinate dehydrogenase to form a TTFA-sensitive succinate-ubiquinone reductase. The maximum reconstitutive activity is 100 mumol of succinate oxidized per min per mg of QPs protein at 23 degrees C. Although cytochrome b560 in isolated QPs is not succinate reducible and its dithionite reduced form is reactive to carbon monoxide, cytochrome b560 is shown to be physically associated with succinate dehydrogenase by the following observations. The dithionite reduced form of cytochrome b560 in isolated QPs has a symmetrical alpha-absorption peak, which upon reconstitution with succinate dehydrogenase becomes slightly broadened and shows a shoulder at around 553 nm, identical to that of cytochrome b560 in succinate-ubiquinone reductase. Upon addition of succinate dehydrogenase to QPs, about 50% of the reduced form of cytochrome b560 in the QPs becomes insensitive to carbon monoxide treatment. The redox potential of cytochrome b560 in QPs is -144 mV which is higher than that of cytochrome b560 in succinate-ubiquinone reductase (-185 mV). Upon addition of succinate dehydrogenase, the redox potential of about 46% of the cytochrome b560 in QPs preparation becomes identical to that of cytochrome b560 in succinate-ubiquinone reductase. Cytochrome b560 in the QPs preparation shows two epr signals, g = 3.07 and g = 2.92, whereas cytochrome b560 in succinate-ubiquinone reductase exhibits only one epr signal at g = 3.46. When QPs is reconstituted with succinate dehydrogenase to form succinate-ubiquinone reductase, the g = 3.46 epr signal reappears at the expense of the g = 3.07 signal. Based on epr measurement at liquid helium temperature, about 18% of the total cytochrome b in the isolated active succinate-cytochrome c reductase is cytochrome b560, indicating that cytochrome b560 is indeed a unique cytochrome b and not a denatured product of cytochrome b562 or b565.
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PMID:Properties of bovine heart mitochondrial cytochrome b560. 302 80

The functional role and topographical orientation in the inner membrane of subunit VII, the ubiquinone-binding protein, of the cytochrome b-c1 complex of yeast mitochondria has been investigated. The apparent molecular weight of this subunit on sodium dodecyl sulfate-urea gels was calculated to be 15,500, while its amino acid composition was similar to that of the Q-binding proteins present in the cytochrome b-c1 complexes isolated from both beef heart and yeast mitochondria. The specific antibody obtained against subunit VII inhibited 30-47% of the ubiquinol-cytochrome c reductase activity in the isolated cytochrome b-c1 complex and in submitochondrial particles but had no effect on cytochrome c reductase activity in mitoplasts, mitochondria from which the outer membrane has been removed. Furthermore, the antibody against subunit VII strongly inhibited (74%) the reduction of cytochrome b by succinate in the presence of antimycin, an inhibitor of center i, but had no effect on cytochrome b reduction in the presence of myxothiazol, an inhibitor of center o. These results suggest that subunit VII, the Q-binding protein, is involved in electron transport at center o of the cytochrome b-c1 complex of the respiratory chain and that subunit VII is localized facing the matrix side of the inner mitochondrial membrane.
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PMID:Subunit VII, the ubiquinone-binding protein, of the cytochrome b-c1 complex of yeast mitochondria is involved in electron transport at center o and faces the matrix side of the membrane. 303 32

The respiratory systems of the mother cells and forespores of Bacillus cereus were compared throughout the maturation stages (III to VI) of sporulation. The results indicated that both cell compartments contain the same assortment of oxidoreductases and cytochromes. However membrane fractions from young forespores were clearly distinct from those of the mother cell, i.e., lower content of cytochrome aa3, lower cytochrome c oxidase activity, higher concentration of cytochrome o, and a lower sensitivity of the respiration to the inhibiting effect of cyanide. This suggests that the cyanide-resistant pathway contributes more importantly to forespore respiratory activity than to activity in the mother cell compartment. During the maturation stages, the forespore NADH oxidase activity declined faster than in the mother cells. Other activities studied decreased steadily in both cell compartments. These findings together with the analysis of the kinetics of NADH-dependent reduction of cytochromes in the mature spore membranes indicated an impairment of electron flow between NADH dehydrogenase and cytochrome b. This impairment could be overcome by the addition of menadione.
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PMID:Respiratory systems of the Bacillus cereus mother cell and forespore. 309 18

Growth of Mycobacterium phlei under low oxygen tension resulted in specific activities two to twenty times lower for formate dehydrogenase, malate dehydrogenase, beta-hydroxybutyrate dehydrogenase, lactate oxidase and NADH dehydrogenase than when cultures were grown under high aeration. An increase in fumarate reductase and succinate dehydrogenase occurred with M. phlei grown under low oxygen tension. Malate: vitamin K dehydrogenase and glucose-6-phosphate dehydrogenase activity were not significantly affected by the oxygen tension used to grow the bacteria, and neither culture contained a lactate dehydrogenase. With growth of M. phlei in conditions of low oxygen tension, cytochrome a was not detected, but cytochrome b was prominent in membranes and cytochrome c was present in the soluble fraction.
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PMID:Influence of oxygen tension on the respiratory activity of Mycobacterium phlei. 318 14

The three E-beta-methoxyacrylate (MOA) inhibitors oudemansin A, strobilurin A and MOA stilbene [3-methoxy-2(2-styrylphenyl)propenic acid-methylester], which differ by more than one order of magnitude in their binding affinity to the mitochondrial ubihydroquinone:cytochrome c oxidoreductase (bc1 complex), bind to a site that is not identical to the binding site for ubihydroquinone, the substrate of the outer ubiquinone reaction site (Qo centre). Although the ubihydroquinone molecule is still bound in the presence of the MOA inhibitors, its electrons cannot be transferred to the iron-sulfur centre. A shift of the relative position of the ubihydroquinone molecule in the reaction centre due to a conformational distortion of cytochrome b induced by the binding of the MOA inhibitor seems to be the reason for the blocked electron transfer. Further analysis shows that ubihydroquinone affects the Kd values of all three MOA inhibitors tested: the values are raised by a constant factor of two, although the inhibitors bind with quite different affinity. The iron-sulfur protein is not involved in the binding of the MOA inhibitors. These results have direct implications for the proper use of MOA inhibitors in experiments designed to analyse the structure/mechanism relationship in cytochrome c reductase. In particular, point mutations recently described in MOA-inhibitor-resistant mutants can no longer be taken to affect necessarily the ubihydroquinone binding site.
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PMID:Characterisation of binding of the methoxyacrylate inhibitors to mitochondrial cytochrome c reductase. 337 45

Functional organization of mitochondrial genome (maxicircle kinetoplast DNA (kDNA)) from a flagellate protozoan Crithidia oncopelti was studied by Northern hybridization. A set of overlapping transcripts were mapped in the conserved region of the maxicircle. Several large (3-4 kb) RNAs, overlapping two or more smaller transcripts were found. Four regions produce a couple of RNAs differing in size 50-100 bases. Southern hybridization with several probes from the maxicircle kDNA of Leishmania tarentolae allowed identification of some of the found transcripts as corresponding to NADH dehydrogenase, subunit IV (Nd IV), cytochrome oxidase, subunits I-II (Cox I-II), cytochrome b (Cyt. b), ORF6-genes. Regions, homologous to the probes used are arranged in the same fashion in C. oncopelti kDNA as related genes in L. tarentolae. The divergent region was proved to be poorly transcribed and to produce a set of RNAs from 0.5 to 2.3 kb. Some transcripts of the divergent region seem to hybridize with distant maxicircular fragments. Cross-hybridization of such fragments has shown the absence of the regions of continuous homology.
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PMID:Transcripts of the maxicircle kinetoplast DNA of Crithidia oncopelti. 343 71

Mitochondria isolated from coenzyme Q deficient yeast cells had no detectable NADH:cytochrome c reductase or succinate:cytochrome c reductase but had comparable amounts of cytochromes b and c1 as wild-type mitochondria. Addition of succinate to the mutant mitochondria resulted in a slight reduction of cytochrome b; however, the subsequent addition of antimycin resulted in a biphasic reduction of cytochrome b, leading to reduction of 68% of the total dithionite-reducible cytochrome b. No "red" shift in the absorption maximum was observed, and no cytochrome c1 was reduced. The addition of either myxothiazol or alkylhydroxynaphthoquinone blocked the reduction of cytochrome b observed with succinate and antimycin, suggesting that the reduction of cytochrome b-562 in the mitochondria lacking coenzyme Q may proceed by a pathway involving cytochrome b at center o where these inhibitors block. Cyanide did not prevent the reduction of cytochrome b by succinate and antimycin the the mutant mitochondria. These results suggest that the succinate dehydrogenase complex can transfer electrons directly to cytochrome b in the absence of coenzyme Q in a reaction that is enhanced by antimycin. Reduced dichlorophenolindophenol (DCIP) acted as an effective bypass of the antimycin block in complex III, resulting in oxygen uptake with succinate in antimycin-treated mitochondria. By contrast, reduced DCIP did not restore oxygen uptake in the mutant mitochondria, suggesting that coenzyme Q is necessary for the bypass. The addition of low concentrations of DCIP to both wild-type and mutant mitochondria reduced with succinate in the presence of antimycin resulted in a rapid oxidation of cytochrome b perhaps by the pathway involving center o, which does not require coenzyme Q.
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PMID:Reduction of cytochrome b in mitochondria from yeast lacking coenzyme Q. 354 40


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