Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:O14944 (EPR)
 13,097 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In 5,5'-dithiobis(2-nitrobenzoate) (DTNB)-treated succinate: cytochrome c reductase, the electron transfer from duroquinol to cytochrome c is inhibited due to the fact that the Rieske Fe-S cluster and, consequently, cytochrome, c, are no longer reducible by substrate. The finding that, after this treatment, cytochrome b is still reducible by substrate in the absence of antimycin, but not in its presence, is consistent with a Q-cycle mechanism for the electron transfer through QH2:cytochrome c oxidoreductase. The inhibitory effect of DTNB and its effect on the EPR spectrum of the [2Fe-2S] cluster suggest that it prevents either the binding of ubiquinone in the vicinity of this cluster or the interaction between the Fe-S protein and a ubiquinone-binding protein.
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PMID:The site of inhibition by 5,5'-dithiobis(2-nitrobenzoate) in ubiquinol: cytochrome c oxidoreductase. 628 87

The pK and mid-point redox potential of the Q-analogue 7-(n-heptadecyl)mercapto-6-hydroxy-5,8-quinolinequinone (HMHQQ) in aqueous medium are so low that under the experimental conditions used for studying the inhibition of electron transfer in submitochondrial particles only the oxidized, anionic form is present. The KD of the analogue, determined by comparing its inhibitory effect with that of n-heptyl-4-hydroxyquinoline N-oxide, is (0.003 + 0.24 x mg protein/ml) microM. The inhibition of succinate oxidation is pH dependent, due to a pH-dependent change in the overcapacity of the QH2-oxidizing system above the Q-reducing system. If the terminal part of the respiratory chain is reduced with ascorbate, the analogue inhibits the reduction of cytochrome b by substrate in the presence of antimycin with a similar KD value. In the absence of ascorbate the KD value is 100-times higher. The reduction of cytochrome b by substrate in particles treated with 2,3-dimercaptopropanol (BAL) + O2 is also sensitive to HMHQQ, with a KD value in between the two values given above. It is concluded that the QH2 oxidase system contains two different sites for interaction with ubiquinone. The site responsible for the inhibition of steady-state electron transfer is near the Fe-S cluster, as is shown by the sensitivity to the redox state of this cluster and by the effect of HMHQQ on the EPR signal of the reduced cluster. The second site, which is similar to the antimycin-binding site, is occupied only at higher concentrations of inhibitor. The affinity of HMHQQ for this site is not affected by the redox state of the Fe-S cluster.
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PMID:Identification of two different Q-binding sites in QH2-cytochrome c oxidoreductase, using the Q analogue n-heptadecylmercapto-6-hydroxy-5,8-quinolinequinone. 629 2

NADH-ubiquinone (Q) reductase isolated from beef heart mitochondria exhibited, upon reduction by NADH, a prominent EPR signal at room temperature attributable to stable ubisemiquinone radical(s). The concentration of the ubisemiquinone radical reached as high as 40% of the total Q content in the reductase. The radical was virtually abolished by adding rotenone, whereas rotenone had no effect on the reduction of FMN by NADH. The radical showed an EPR signal of g = 2.0042 at approximately 9.5 GHz with no resolved hyperfine structure and had a line width of 6.8 Gauss at 23 degrees C. The Q-band EPR spectra at 35 GHz showed well resolved g-anisotropy and had a field separation between derivative extrema of 24 Gauss. These results substantiate the fact that this radical was bound to a protein; we call it ubiquinone protein-N (QP-N). The pH dependence of the EPR signals demonstrated that the species of the ubisemiquinone radical(s) consisted of not only an anionic form but also a neutral form. Only about half of the QP-N radical formed by NADH reduction was abolished by p-chloromercuric sulfonate. The microwave power saturation curve of the radical was biphasic; the first phase leveled off at about 5 milliwatts and then at about 20 milliwatts. These results suggested that the ubisemiquinone radical from QP-N was heterogenous, consisting of at least two populations of stable ubisemiquinone radical(s). It is suggested that two kinds of QP-N exist in NADH-Q reductase. Each mole of protein may bind two mol of Q.
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PMID:Evidence of an ubisemiquinone radical(s) from the NADH-ubiquinone reductase of the mitochondrial respiratory chain. 629 5

3-Alkyl-2-hydroxy-1,4-naphthoquinones (alkyl-HNQ) inhibit Rieske iron-sulfur cluster (Rieske FeS) oxidation and cytochrome b reduction in ubiquinol-cytochrome c oxidoreductase. The effects are the same as those of 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole. Concentrations for 50% inhibition in chromatophores of Rhodopseudomonas sphaeroides (at 0.4 microM reaction center) are 2 microM for undecyl-, 3 microM for octyl-, and 40 microM for pentyl-substituted hydroxynaphthoquinones. The ethyl-substituted and unsubstituted derivatives do not inhibit electron transfer below 2 mM. In chromatophores in which the ubiquinone is partially extracted by isooctane (leaving 4 ubiquinones/reaction center), undecyl-HNQ is effective at 2.5 times lower concentration than in normal chromatophores (30 ubiquinones/reaction center). This observation suggests that the binding of the inhibitor is competitive with ubiquinone. Undecyl-HNQ eliminates the effect that the ubiquinone redox state has on the line shape of the EPR signal of Rieske FeS. This supports the idea that alkyl-HNQ shares a common binding site with ubiquinone which is closely associated with Rieske FeS. The ubiquinone in question has a midpoint oxidation-reduction potential at pH 7 of 90 mV with a -60 mV/pH unit dependency. This value matches that of the ubiquinone pool rather than that of ubiquinone Z, which is functionally recognized as a component "between" cytochrome b and Rieske FeS. When Rieske FeS is oxidized, a 20 times higher concentration of undecyl-HNQ is required for the electron transfer inhibition. This is consistent with the observation that the binding of the inhibitor shifts the midpoint oxidation-reduction potential of Rieske FeS about 60 mV higher, which in turn means that the inhibitor binds about 10 times stronger to the site when Rieske FeS is reduced than when it is oxidized. The observations suggest that 3-alkyl-2-hydroxy-1,4-naphthoquinones inhibit electron transfer by acting as ubiquinone antagonists at a site closely associated with Rieske FeS.
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PMID:Inhibition of electron transfer by 3-alkyl-2-hydroxy-1,4-naphthoquinones in the ubiquinol-cytochrome c oxidoreductases of Rhodopseudomonas sphaeroides and mammalian mitochondria. Interaction with a ubiquinone-binding site and the Rieske iron-sulfur cluster. 629 6

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

The antimycin-sensitive ubisemiquinone radical (QC) of the ubiquinol-cytochrome c oxidoreductase of submitochondrial particles and chromatophores of Rhodopseudomonas sphaeroides Ga has been studied by a combination of redox potentiometry and EPR spectroscopy. This g = 2.005 radical signal appears at physiological pH values and increases in intensity with increasing pH up to pH 7.6 in submitochondrial particles and pH 9.0 in R. sphaeroides after which its intensity remains unchanged. The Em7 (ubiquinone/quinol) of the signal, estimated from redox titration data is 80 mV for submitochondrial particles, and 150 mV in chromatophores. Each of these values is higher than that of the quinone pool by 20 mV in submitochondrial particles and 60 mV in R. sphaeroides. This indicates that the quinone at the binding site is out of equilibrium with the pool, and that binding site preferentially binds quinol over quinone. Analysis of the shapes of the semiquinone titration curves, taken together with the midpoint elevation, indicates a quinone-binding site: cytochrome c1 stoichiometry of 1:1 in both submitochondrial particles and chromatophores. At its maximal intensity, the semiquinone concentration at the binding site is 0.26 in submitochondrial particles (greater than pH 7.6) and 0.4 in chromatophores (greater than pH 9.0). In both systems, the midpoint of the ubiquinone/ubisemiquinone couple is constant as the pH is raised up to the pH of maximal semiquinone formation whereafter it becomes more negative at the rate of -60 mV/pH unit. The midpoint of the ubisemiquinone/quinol couple, on the other hand, varies by -120 mV/pH unit at pH values up to the transition pH, after which it, too, changes by -60 mV/pH unit. This seemingly anomalous behavior may be explained by invoking a protonated group at or near the quinone-binding site whose pK corresponds to the pH transition point in the quinone/semiquinone/quinol redox chemistry when the site is free or when quinone or quinol occupies the site. This pK is elevated to at least pH 9.0 in submitochondrial particles and 10.5 in R. sphaeroides when semiquinone is bound to the site.
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PMID:Thermodynamic properties of the semiquinone and its binding site in the ubiquinol-cytochrome c (c2) oxidoreductase of respiratory and photosynthetic systems. 631 10

From a study of the magnetic field dependence of the linear electric field effect (LEFE) in EPR spectroscopy, we demonstrate that iron-sulfur cluster 3 in air-oxidized, beef heart succinate-ubiquinone oxidoreductase (Complex II) is a 3-iron cluster. This suggests that cluster 3 may arise by oxidative degradation from a 4-iron cluster originally present in the enzyme and may be reconverted back into a 4-iron cluster under reducing conditions. Linear electric field effect studies of succinate-reduced Complex II are in accord with the view that cluster 1 is a 2-iron cluster.
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PMID:Iron-sulfur cluster 3 of beef heart succinate-ubiquinone oxidoreductase is a 3-iron cluster. 632 51

Reaction of ubiquinone in the high-affinity quinone-binding site (QH) in bo-type ubiquinol oxidase from Escherichia coli was revealed by EPR and optical studies. In the QH site, ubiquinol was shown to be oxidized to ubisemiquinone and to ubiquinone, while no semiquinone signal was detected in the oxidase isolated from mutant cells that cannot synthesize ubiquinone. The QH site highly stabilized ubisemiquinone radical with a stability constant of 1-4 at pH 8.5 and the stability became lower at the lower pH. Midpoint potential of QH2/Q couple was -2 mV at pH 8.5 and showed -60 mV/pH dependence indicative of 2H+/2e- reaction. The Em was more negative than that of low-spin heme b above pH 7.0. We conclude that the QH mediates intramolecular electron transfer from ubiquinol in the low-affinity quinol oxidation site (QL) to low-spin heme b. Unique roles of the quinone-binding sites in the bacterial ubiquinol oxidase are discussed.
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PMID:Stabilization of a semiquinone radical at the high-affinity quinone-binding site (QH) of the Escherichia coli bo-type ubiquinol oxidase. 758 50

The proton-translocating NADH:ubiquinone oxidoreductase (complex I) was isolated from Escherichia coli by chromatographic steps performed in the presence of an alkylglucoside detergent at pH 6.0. The complex is obtained in a monodisperse state with a molecular mass of approximately 550,000 Da and is composed of 14 subunits. The subunits were assigned to the 14 genes of the nuo operon, partly based on their N-terminal sequences and partly on their apparent molecular masses. The preparation contains one noncovalently bound FMN/molecule. At least two binuclear (N1b and N1c) and three tetranuclear (N2, N3 and N4) iron-sulfur clusters were detected by EPR in the preparation when reduced with NADH. Their EPR characteristics remained mostly unaltered during the isolation process. After reconstitution in phospholipid membranes, the preparation catalyses piericidin-A-sensitive electron transfer from NADH to ubiquinone-2 with Km values similar to those of complex I in cytoplasmic membranes but with only 10% of the Vmax value. The isolated complex I was cleaved into three fragments when the pH was raised from 6.0 to 7.5 and the detergent exchanged to Triton X-100. One of these fragments is a water-soluble NADH dehydrogenase fragment which is composed of three subunits bearing at least four iron-sulfur clusters (N1b, N1c, N3 and N4) that can be reduced with NADH, one of them bearing FMN. The second, amphipathic, fragment, which is presumed to connect the NADH dehydrogenase fragment with the membrane, contains four subunits and at least one EPR-detectable iron-sulfur cluster whose spectral properties are reminiscent of the eucaryotic cluster N2. The third membrane fragment is composed of seven homologues of the mitochondrially encoded subunits of the eucaryotic complex I. This subunit arrangement coincidences to some extent with the order of the genes on the nuo operon. A topological model of the E. coli complex I is proposed.
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PMID:Isolation and characterization of the proton-translocating NADH: ubiquinone oxidoreductase from Escherichia coli. 760 27

The properties of the quinone acceptor complex in the photosynthetic reaction center of the atrazine-resistant Rhodopseudomonas viridis mutant A2 (Glu L212-->Lys) were studied by EPR spectroscopy and by photoelectric measurements. The EPR signal attributed to the semiquinone-iron (QB-Fe2+) was significantly different from wild type and resembled that found in PS II. Essentially normal oscillations of QB-Fe2+ were observed upon flash illumination. The kinetics of the first and the second electron transfer from QA to QB were characterized by a photoelectric double-flash method. Compared to wild type, the rate of the first electron transfer in the large majority of reaction centers was decreased drastically from k1 = (18 microseconds)-1 in the wild type to (70 ms)-1 in the mutant, whereas the second electron transfer was only slightly slowed down with a rate of k2 = (260 microseconds)-1 compared to (65 microseconds)-1 in wild type (pH 7). When the pH was raised above 10, in a major fraction of the reaction centers a fast kinetics of the first electron transfer, like that in wild type, reappeared. The experimental results are interpreted as an effect of the positive charge on the lysine causing a significant structural change of the QB binding pocket and a strongly diminished affinity for ubiquinone. The slow QA(-)-->QB electron transfer kinetics are thus attributed to ubiquinone binding, which is rate limiting. The possible role of the residue Glu L212, which is conserved in all purple bacteria, in electron and proton transfer to QB is discussed.
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PMID:Structural and functional consequences of a Glu L212-->Lys mutation in the QB binding site of the photosynthetic reaction center of Rhodopseudomonas viridis. 772 86


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