UNIPROT:O14944 - FACTA Search

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Query: UNIPROT:O14944 (EPR)
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Potentiometric measurements have been performed on Complex III from bakers' yeast. The midpoint potentials for the b and c cytochromes were measured using room-temperature MCD and liquid-helium temperature EPR. A value of 270 mV was obtained for cytochrome c1, regardless of temperature, while the midpoint potentials found for the two species of cytochrome b varied with temperatures, viz., 62 and -20 mV at room temperature (MCD) compared to 116 and -4 mV at about 10 K (EPR). The midpoint potential of the iron-sulfur center obtained by low-temperature EPR was 286 mV. An abrupt conformational change occurred immediately after this center was fully reduced resulting in a change in EPR line shape. The potentials of the two half-reactions of ubiquinone were measured by following the semiquinone radical signal at 110 K and 23 degrees C. Potentials of 176 and 51 mV were found at low temperature, while values of 200 and 110 mV were observed at room temperature. The midpoint potential of cytochrome c1 was found to be pH independent. The potentials of cytochrome b were also independent of pH when titrations were performed in deoxycholate buffers, while a variation of -30 mV per pH unit was observed for both cytochrome c species in taurocholate buffers. These two detergents also produced different MCD contributions of the two b cytochromes. A decrease in Em of greater than 300 mV was found in potentiometric measurements of cytochrome c1 at high ratios of dye to Complex III. Antimycin does not affect the redox potentials of cytochrome c1 but appears to induce a transition of the low-potential b heme to a high-potential species. This transition is mediated by ubiquinone.
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PMID:Potentiometric studies on yeast complex III. 630 54

Myxothiazol, an antibiotic from Myxococcus fulvus, which inhibits mitochondrial respiration in the bc1 complex of the respiratory chain, has effects on the redox components of isolated succinate-cytochrome c reductase complex which suggest that it interacts with both cytochrome b and the iron-sulfur protein of the bc1 complex. The inhibitor appears to increase the midpoint potentials of cytochromes b-562 and b-566, as indicated by an increase in their reducibility by the succinate/fumarate couple. It also causes a red shift in the optical spectrum of ferrocytochrome b-566, as reported previously (Becker, W. F., Von Jagow , G., Anke , T., Steglisch , W. (1981) FEBS Lett. 132, 329-333). This red shift is enhanced by Triton X-100, and there is no shift in the spectrum of b-562. These results are consistent with evidence that mutations conferring myxothiazol resistance in yeast map to the mitochondrial gene for cytochrome b ( Thierbach , G., and Michaelis, G. (1982) Mol. Gen. Genet. 186, 501-506). In addition, myxothiazol has effects on reduction of the cytochromes b and c1 by succinate or ubiquinol which are identical to those caused by removal of the iron-sulfur protein from the bc1 complex. It blocks reduction of cytochrome c1 during single and multiple turnovers of the bc1 complex, but does not block reduction of the b cytochromes. In the presence of antimycin, it blocks reduction of both cytochromes b and c1. In contrast to antimycin, myxothiazol inhibits oxidant-induced reduction of both b cytochromes and does not inhibit their oxidation by fumarate. Myxothiazol also inhibits reduction of the iron-sulfur protein by ubiquinol and shifts the gx resonance in the EPR spectrum of the iron-sulfur protein from g = 1.79 to 1.76. It does not affect the midpoint potential of the iron-sulfur protein, but does eliminate the increase in midpoint potential which is caused by inhibitory hydroxyquinones which bind to the iron-sulfur protein. The effects of myxothiazol are consistent with a protonmotive Q cycle pathway of electron transfer in which myxothiazol binds to cytochrome b and displaces quinone from the iron-sulfur protein of the bc1 complex. These results suggest either that a myxothiazol-induced conformational change in cytochrome b is transmitted to a quinone binding site on the iron-sulfur protein, or that there is a quinone binding site which consists of peptide domains from both cytochrome b and iron-sulfur protein.
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PMID:An inhibitor of mitochondrial respiration which binds to cytochrome b and displaces quinone from the iron-sulfur protein of the cytochrome bc1 complex. 632 77

The Escherichia coli quinol oxidase, cytochrome bo, is closely related to the cytochrome c oxidase, cytochrome aa3 in all aspects of its structure and function except for the replacement of the cytochrome-c-binding site and its attendant CuA prosthetic group with a quinone-binding site. The putative oxidation of quinol by ferrihaem (cytochrome b) at this site in sequential one-electron steps requires the stabilisation of semiquinone. We have observed, by electron paramagnetic resonance, the properties of a ubisemiquinone radical in appropriately poised samples of purified enzyme reconstituted with excess ubiquinone. The ubisemiquinone is highly stabilised with respect to free ubisemiquinone; significant free radical can be observed even at pH 7.0, while at pH 9.0 the stability constant is 5-10. The pH dependence of the stability constant indicates that the anionic form of the semiquinone predominates above pH 7.5. The two-electron couple has an Em7 of approximately 70 mV. Below pH 9, the pH dependence of the two-electron couple is -60mV/pH, indicative of a 2H+/2e- reaction. The line width of the EPR spectrum is approximately 0.9 mT, which is consistent with a ubisemiquinone anion. In comparison with other respiratory chain Q.- species that have been described, the relaxation rate in the presence of reduced haems appears comparable to magnetically isolated Q.- radicals. Partially resolved splittings of approximately 0.4 mT can be observed in the spectrum of Q.-bo (QH.bo).
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PMID:Studies on a stabilisation of ubisemiquinone by Escherichia coli quinol oxidase, cytochrome bo. 786 53

The nitric oxide reductase (NOR) from Pseudomonas stutzeri is a cytochrome bc complex which shows on SDS/PAGE two subunits with apparent molecular masses of 17 kDa and 38 kDa. Two other species of approximately 45 kDa and 74-78 kDa represent the undissociated enzyme complex and an aggregate of the cytochrome b subunit, respectively. The cytochrome b subunit is highly hydrophobic and results in aberrant electrophoretic mobility. The stability of the enzyme in various detergents and at different pH was investigated. The highest specific activity of 60 mumol NO min-1 mg-1 protein was obtained after electrophoresis in the presence of laurylpropanediol-3-phosphorylcholine ether. Purified NOR contained cardiolipin, phosphatidylglycerol, and phosphatidylethanolamine, the latter as the major component. A phospholipid was required for high catalytic activity with either cardiolipin or phosphatidylglycerol increasing the activity of the enzyme as isolated by a factor of up to 5. Free fatty acids inhibited NOR, with cis-9-octadecenoic acid (oleic acid) showing the most pronounced effect. Certain detergents substituted for the phospholipid requirement of NOR. The enzyme, as isolated, in 0.1% Triton X-100, 20 mM Tris/HCl pH 8.5, exhibited a complex set of EPR resonances at low magnetic field, with a prominent peak at g 6.34 resulting from Fe(III) high-spin cytochrome b. The second prominent feature arose from a low-spin Fe(III) heme center with strong lines at apparent g values of 3.02 and 2.29, and a broad resonance at g approximately 1.5 which we assigned to the cytochrome c component of the enzyme. From spin quantitation and computer simulations of the various EPR signals a ratio close to 1:1 for the low-spin/high-spin heme centers in NOR was estimated. Shifting the pH from 8.5 to 5.0, replacing Triton X-100 by other detergents, or adding soybean phospholipids to the protein, led to pronounced changes of the EPR signals in the g = 6 region. In contrast, the strong inhibitor oleic acid did not cause significant spectral changes. NOR which had been reduced by L-ascorbate/phenazine methosulfate prior to incubation with its substrate NO gave the characteristic Fe(II) nitrosyl triplet centered at g approximately 2.01, with a hyperfine splitting of 1.70 mT. In the absence of dioxygen, NOR was quantitatively reduced by either sodium dithionite, or photochemically with deazaflavin and oxalate; the enzyme was reoxidizable by ferricyanide in a fully reversible reaction. Spectroelectrochemical oxidoreductive titrations gave E'o (versus standard hydrogen electrode) = +322 mV for the cytochrome b and +280 mV for the cytochrome c component.
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PMID:Nitric oxide reductase from Pseudomonas stutzeri, a novel cytochrome bc complex. Phospholipid requirement, electron paramagnetic resonance and redox properties. 802 Apr 68

The Rieske iron-sulfur subunit of the cytochrome bc1 complex from Rhodobacter sphaeroides has been expressed in Escherichia coli and also in a strain of Rb. sphaeroides lacking the other subunits of the bc1 complex. PCR products encoding the full-length subunit were introduced into expression vectors to produce the subunit alone or the subunit fused behind the mature portion of the E. coli maltose binding protein (MBP), but lacking the MBP signal sequence. These proteins are both located in the cytoplasmic membrane. The unfused Rieske subunit assembles a Rieske-like iron-sulfur cluster, but with EPR characteristics which differ from the normal rhombic signal observed in the cytochrome bc1 complex. The overproduced MBP fusion protein, on the other hand, does not contain an EPR-detectable iron-sulfur cluster. Subfragments of the Rieske subunit lacking the amino-terminal hydrophobic anchor also lack the iron-sulfur cluster were expressed in E. coli. When expressed in Rb. sphaeroides in the absence of the cytochrome b and c1 subunits, the fully metalated Rieske subunit with the diagnostic gy = 1.90 EPR signal is observed in the cytoplasmic membrane. The fact that the Rieske subunit has an assembled iron-sulfur cluster and is bound to either the E. coli or the Rb. sphaeroides membrane in the absence of the other subunits of the bc1 complex demonstrates a mode of membrane attachment independent of the other components of the complex. These data are consistent with models in which the Rieske subunit is bound to the membrane via a single membrane-spanning helix located near the amino terminus.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Assembly of the Rieske iron-sulfur subunit of the cytochrome bc1 complex in the Escherichia coli and Rhodobacter sphaeroides membranes independent of the cytochrome b and c1 subunits. 838 Jul 4

Deletion of QCR9, the nuclear gene encoding the 7.3-kDa subunit 9 of the cytochrome bc1 complex, impairs respiration of Saccharomyces cerevisiae, coincident with loss of ubiquinol-cytochrome c oxidoreductase activity. Optical spectra of mitochondrial membranes from yeast in which the gene for subunit 9 is deleted show a diminution of cytochrome b absorption similar to the spectra of membranes from yeast in which the gene for the Rieske iron-sulfur protein is deleted, suggesting an interaction between subunit 9, iron-sulfur protein, and cytochrome b. Synthesis of cytochrome b by mitochondria from the deletion strain is unimpaired, indicating that the diminished b absorption is due to a post-assembly effect on the heme environment resulting from the absence of subunit 9. Iron-sulfur protein is present in normal amounts and processed to its mature form in the absence of subunit 9, although the protein is more labile to endogenous proteases during the isolation of membranes. EPR spectroscopy of membranes from the subunit 9 deletion strain indicates that the g = 1.90 signal characteristic of the Rieske iron-sulfur cluster is absent, even though mature sized apoprotein is present. Pre-steady state reduction of cytochrome c1 is markedly slowed, but not eliminated, in the subunit 9 deletion strain, which suggests that an EPR-silent, sluggishly reactive derivative of the iron-sulfur cluster is present. These results suggest that in the absence of subunit 9 the conformation of iron-sulfur protein is altered such that the protein is more labile, the iron-sulfur cluster is not properly inserted, and iron-sulfur protein interaction with cytochrome b is modified in a manner which distorts the heme environment. This is the first instance in which deletion of one of the supernumerary subunits of the cytochrome bc1 complex results in the loss of function of a redox center within the complex, without a concomitant loss of other subunits.
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PMID:Subunit 9 of the Saccharomyces cerevisiae cytochrome bc1 complex is required for insertion of EPR-detectable iron-sulfur cluster into the Rieske iron-sulfur protein. 838 62

A simple system for aerobic assay of the quinol-fumarate reductase reaction catalyzed by purified soluble bovine heart succinate-ubiquinone reductase in the presence of NADH, NAD(P)H-quinone reductase (DT-diaphorase) and an appropriate quinone is described. The reaction is inhibited by carboxin, suggesting that the same quinone/quinol binding site is involved in electron transfer from succinate to ubiquinone and from ubiquinol to fumarate. The kinetic properties of the reaction in both directions and comparative affinities of the substrate binding sites of the enzyme to substrates (products) and competitive inhibitors are reported. Considerable difference in affinity of the substrates binding site to oxaloacetate was demonstrated when the enzyme was assayed in the direct and reverse directions. These results were taken to indicate that the oxidized dicarboxylate-free enzyme is an intermediate during the steady-state succinate-ubiquinone reductase reaction, whereas the reduced dicarboxylate-free enzyme is an intermediate of the steady-state ubiquinol-fumarate reductase reaction. No difference in the reactivity of the substrate-protected cysteine and arginine residues was found when the pseudo-first-order rate constants for N-ethylmaleimide and phenylglyoxal inhibition were determined for oxidized and quinol-reduced enzyme. Quinol-fumarate reductase activity was reconstituted from the soluble succinate dehydrogenase and low-molecular-mass ubiquinone reactivity conferring protein(s). No reduction of cytochrome b was observed in the presence of quinol generating system, whereas S-3 low temperature EPR-detectable iron-sulfur center was completely reduced by quinol under equilibrium (without fumarate) or steady-state (in the presence of fumarate). No significant reduction of ferredoxin type iron-sulfur centers was detected during the steady-state quinol-fumarate oxidoreductase reaction. The data obtained eliminate participation of cytochrome b in the quinol-fumarate reductase reaction and show that the rate limiting step of the overall reaction lies between iron-sulfur center S-3 and lower midpoint potential redox components of the enzyme.
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PMID:Fumarate reductase activity of bovine heart succinate-ubiquinone reductase. New assay system and overall properties of the reaction. 841 79

Inhibition of photosystem II electron transport by UV-B radiation has been studied in isolated spinach photosystem II membrane particles using low-temperature EPR spectroscopy and chlorophyll fluorescence measurements. UV-B irradiation results in the rapid inhibition of oxygen evolution and the decline of variable chlorophyll fluorescence. These effects are accompanied by the loss of the multiline EPR signal arising from the S2 state of the water-oxidizing complex and the induction of Signal IIfast originating from stabilized Try-Z+. The EPR signals from the QA-Fe2+ acceptor complex, Tyr-D+, and the oxidized non-heme iron (Fe3+) are also decreased during the course of UV-B irradiation, but at a significantly slower rate than oxygen evolution and the multiline signal. The decrease of the Fe3+ signal at high g values (g = 8.06, g = 5.6) is accompanied by the induction of another EPR signal at g = 4.26 that arises most likely from the same Fe3+ ion in a modified ligand environment. UV-B irradiation also affects cytochrome b-559. The g = 2.94 EPR signal that arises from the dark- oxidized form is enhanced, whereas the light inducible g = 3.04 signal that arises from the photo-oxidizable population of cytochrome b-559 is diminished. UV-B irradiation also induces the degradation of the D1 reaction center protein. The rate of the D1 protein loss is slower than the inhibition of oxygen evolution and of the multiline signal but follows closely the loss of Signal IIslow, the QA-Fe2+ and the Fe3+ EPR signals, as well as the release of protein-bound manganese. It is concluded from the results that UV-B radiation affects photosystem II redox components at both the donor and acceptor side. The primary damage occurs at the water-oxidizing complex. Modification and/or inactivation of tyrosine-D, cytochrome b-559, and the QAFe2+ acceptor complex are subsequent events that coincide more closely with the UV-B-induced damage to the protein structure of the photosystem II reaction center.
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PMID:UV-B-induced inhibition of photosystem II electron transport studied by EPR and chlorophyll fluorescence. Impairment of donor and acceptor side components. 868 33

o-Phenanthroline and m-phenanthroline both inhibit the electron transfer activity of lauryl maltoside-solubilized yeast bc1 complex progressively with time. Pre-steady-state kinetics indicate that these compounds bind to the complex on the intermembrane space side, thereby blocking reduction of cytochrome b via the ubiquinol oxidation site. o-Phenanthroline is additionally capable of chelating an iron atom derived from the Rieske Fe-S cluster, thereby distorting the structure of the Rieske protein. EPR analysis shows that the secondary effect of o-phenanthroline occurs after initial inactivation and that m-phenanthroline, which lacks chelating activity, does not affect the Rieske Fe-S cluster. Spectral analysis shows that the b and c1 cytochromes are still dithionite-reducible after inactivation by o-phenanthroline, indicating that they remain intact. Inactivation by o-phenanthroline can be prevented by the addition of Fe2+. Surprisingly, ferroin, the o-phenanthroline-ferrous sulfate complex, also inhibits the bc1 complex activity. In contrast to o-phenanthroline, this effect is instantaneous. The two types of inhibition are clearly distinguishable by pre-steady-state reduction kinetics. Interestingly, ferroin can only inhibit electron transfer activity by about 50%. This behavior is discussed in relation to the dimeric structure of the bc1 complex, and we conclude that ferroin binds to only one of the two protomers. The rate of inactivation by o-phenanthroline is dependent on the incubation temperature and can be quantitated in terms of the half-life for a certain temperature, the time at which the bc1 activity is reduced to 50%. In contrast to the solubilized form, the bc1 complex in intact mitochondria is insensitive to o-phenanthroline, suggesting that the inactivation rate by o-phenanthroline is dependent on accessibility of the complex to the agent. Reaction with o-phenanthroline is thus a useful technique for study of structural stability of the bc1 complex under different conditions and should provide a sensitive tool for determination of the relative stability of mutant enzymes.
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PMID:Differential inhibition of the yeast bc1 complex by phenanthrolines and ferroin. Implications for structure and catalytic mechanism. 920 79

We have established a new purification procedure of cytochrome b561 from bovine adrenomedullary chromaffin vesicles. The heme content analysis of the purified sample indicated the presence of 1.7 molecules of heme B/cytochrome b561 molecule. EPR spectroscopy of the purified enzyme in oxidized state showed that there were three types of low spin heme species. Two of them showed usual EPR signals at gz = 3.14 and gz = 2.84 arising from the same heme and were interconvertible depending on pH. The other species showed a highly anisotropic low spin signal at gz = 3.70, with a lower redox potential than the others, and a temperature-sensitive character. These properties are very similar to low potential cytochrome b (bL or b566) of the mitochondrial complex III, indicating that the gz = 3.70 species is derived from a heme component different from the one that shows the usual low spin EPR signals. Based on our new structural model, these two heme B prosthetic groups are likely to be located on both sides of the membranes in close contact with the ascorbic acid- and semidehydroascorbic acid-binding sites, respectively, to facilitate the electron transfer across the membranes. This molecular architecture may provide a structural basis for the transmembrane electron transfer catalyzed by this hemoprotein.
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PMID:Existence of two heme B centers in cytochrome b561 from bovine adrenal chromaffin vesicles as revealed by a new purification procedure and EPR spectroscopy. 928 27

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