Mucidin and strobilurin A are identical and inhibit electron transfer in the cytochrome bc1 complex of the mitochondrial respiratory chain at the same site as myxothiazol

Jagow, Gebhard von; Gribble, Gordon W.; Trumpower, Bernard L.

doi:10.1021/bi00352a006

Cited by 76 publications

(39 citation statements)

References 32 publications

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“…A structure-based patent search revealed that the alkyliminophenylacetate compounds contain the toxophores of the strobilurin fungicides (32,33). Azoxystrobin inhibits electron transfer in the cytochrome bc1 complex of the mitochondrial respiratory chain before cytochrome b, resulting in the preventing cytochrome b reduction and thereby attenuating ROS production (35,36). A recent study indicates that Qo site inhibitors of complex III (myxothiazol, stigmatellin) are most effective in preventing hypoxia-induced ROS production and HIF-1 activation.…”

Section: Discussionmentioning

confidence: 99%

A chemical genomics screen highlights the essential role of mitochondria in HIF-1 regulation

Lin¹,

David

Donnelly

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

103

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Hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development and progression by regulating genes that are vital for proliferation, glycolysis, angiogenesis, and metastasis. To identify strategies of targeting the HIF-1 pathway, we screened a siRNA library against the entire druggable genome and a small-molecule library consisting of 691,200 compounds using a HIF-1 reporter cell line. Although the siRNA library screen failed to reveal any druggable targets, the small-molecule library screen identified a class of alkyliminophenylacetate compounds that inhibit hypoxia-induced HIF-1 reporter activity at single-digit nanomolar concentrations. These compounds were found to inhibit hypoxia but not deferoxamine-induced HIF-1␣ protein stabilization. Further analysis indicated that the alkyliminophenylacetate compounds likely inhibit the HIF-1 pathway through blocking the hypoxia-induced mitochondrial reactive oxygen species (ROS) production. Strikingly, all of the nonalkyliminophenylacetate HIF-1 inhibitors identified from the small-molecule library screen were also found to target mitochondria like the alkyliminophenylacetate compounds. The exclusive enrichment of mitochondria inhibitors from a library of >600,000 diverse compounds by using the HIF-1 reporter assay highlights the essential role of mitochondria in HIF-1 regulation. These results also suggest that targeting mitochondrial ROS production might be a highly effective way of blocking HIF-1 activity in tumors.hypoxia-inducible factor ͉ hypoxia ͉ reactive oxygen species ͉ siRNA ͉ RNAi H ypoxia-inducible factor-1 (HIF-1) is the master regulator of cellular responses to low oxygen (1-3). It consists of a constitutively expressed ␤ subunit and an oxygen-regulated ␣ subunit. The proline hydroxylase domain-containing proteins (PHDs) were originally thought to be the main oxygen sensor that mediates oxygen-dependent HIF-1␣ degradation. Under aerobic conditions, PHDs hydroxylate HIF-1␣ at key proline residues by using oxygen as a substrate, which ultimately causes polyubiquitination and degradation of HIF-1␣ through an E3 ubiquitin ligase complex containing the von Hippel-Lindau protein (4, 5). Because oxygen is required for PHD-mediated hydroxylation, the low oxygen level under hypoxic conditions prevents HIF-1␣ hydroxylation, which subsequently leads to the stabilization of HIF-1␣. However, several recent studies demonstrate that the hypoxia-induced production of reactive oxygen species (ROS) in mitochondria is both necessary and sufficient for hypoxia-dependent HIF-1␣ accumulation, suggesting that mitochondria may act as an oxygen sensor for HIF-1␣ regulation by generating ROS under hypoxic conditions (6-9).HIF-1 has emerged as an attractive target for cancer therapy in the last several years. The requirement of HIF-1 for tumor growth has been examined by abrogating the HIF-1 pathway in tumors by using RNAi, small molecule inhibitors, or genetic alterations. Although the degree of tumor responses to HIF-1 inhibition varies among studies, the major...

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Section: Discussionmentioning

confidence: 99%

A chemical genomics screen highlights the essential role of mitochondria in HIF-1 regulation

Lin¹,

David

Donnelly

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

103

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“…rotenone for Complex I, carboxin for Complex II, mucidin for Complex III, and KCN for Complex IV. The choice of mucidin was dictated by its specific inhibition of the steady-state activity of Complex III, resulting in a hyperbolic relationship between activity and the amount of inhibitor (30). It has long been observed that titration with the commonly used antimycin A gives a non-linear inhibition of ubiquinol-cytochrome c oxidoreductase due to the functional asymmetry in the bc 1 complex dimer and to a stimulatory effect upon binding of one molecule of antimycin per dimer at low inhibitor/enzyme ratios (31,32), whereas myxothiazol can also inhibit Complex I (33).…”

Section: Methodsmentioning

confidence: 99%

“…It has long been observed that titration with the commonly used antimycin A gives a non-linear inhibition of ubiquinol-cytochrome c oxidoreductase due to the functional asymmetry in the bc 1 complex dimer and to a stimulatory effect upon binding of one molecule of antimycin per dimer at low inhibitor/enzyme ratios (31,32), whereas myxothiazol can also inhibit Complex I (33). The concentration of rotenone and mucidin, as ethanolic stock solutions, was checked spectrophotometrically (30,34). The inhibition curves were obtained with a non-linear regression fitting procedure performed on all the raw titration values of every set of experimental data using the application program SigmaPlot (35).…”

Section: Methodsmentioning

confidence: 99%

The Mitochondrial Respiratory Chain Is Partially Organized in a Supercomplex Assembly

Bianchi¹,

Genova²,

Castelli

et al. 2004

Journal of Biological Chemistry

252

169

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The model of the respiratory chain in which the enzyme complexes are independently embedded in the lipid bilayer of the inner mitochondrial membrane and connected by randomly diffusing coenzyme Q and cytochrome c is mostly favored. However, multicomplex units can be isolated from mammalian mitochondria, suggesting a model based on direct electron channeling between complexes. Kinetic testing using metabolic flux control analysis can discriminate between the two models: the former model implies that each enzyme may be rate-controlling to a different extent, whereas in the latter, the whole metabolic pathway would behave as a single supercomplex and inhibition of any one of its components would elicit the same flux control. In particular, in the absence of other components of the oxidative phosphorylation apparatus (i.e. ATP synthase, membrane potential, carriers), the existence of a supercomplex would elicit a flux control coefficient near unity for each respiratory complex, and the sum of all coefficients would be well above unity. Using bovine heart mitochondria and submitochondrial particles devoid of substrate permeability barriers, we investigated the flux control coefficients of the complexes involved in aerobic NADH oxidation (I, III, IV) and in succinate oxidation (II, III, IV). Both Complexes I and III were found to be highly rate-controlling over NADH oxidation, a strong kinetic evidence suggesting the existence of functionally relevant association between the two complexes, whereas Complex IV appears randomly distributed. Moreover, we show that Complex II is fully rate-limiting for succinate oxidation, clearly indicating the absence of substrate channeling toward Complexes III and IV.Considerable information exists on the structure at atomic resolution of most of the transmembrane protein complexes forming the mitochondrial respiratory chain; there is, however, still little direct information on the supramolecular organization of the enzymatic complexes in the inner mitochondrial membrane. Two extreme models for their arrangement in the membrane are conceivable: the model of a random organization of the individual respiratory complexes and that of a supercomplex assembly formed by stable association between proteins.The original solid-state model of Chance and Williams (1) changed gradually because the oxidative phosphorylation enzymes were found functionally active when isolated as individual complexes (2) Despite the acceptance of the idea that electron transfer in mitochondrial membranes depends on random collisions between small diffusing molecules (coenzyme Q and cytochrome c) and complexes

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“…The spectra were recorded at 15 K and the spectrometer settings were as follows: microwave power, 15 mw; modulation amplitude, 10-3 Tesla; microwave frequency, 9.45 GHz; modulation frequency, 100 kHz; scan rate, 25 mT/min; protein concentration, 70 mg ml-' (further details in "Materials and Methods"). (29).…”

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confidence: 99%

Functional Characterization and Partial Purification of the Ubiquinol-Cytochrome c Oxidoreductase from Higher Plant Mitochondria (Helianthus tuberosus)

Esposti¹,

Flamini²,

Zannoni³

1985

Plant Physiol.

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The functional and thermodynamic characteristics of the ubiquinolcytochrome (Cyt) c oxidoreductase in a Cyt b/c,-enriched fraction (defined S-1) isolated from Jerusalem artichoke mitochondria (JAM) (Helianthus tuberosus), have been analyzed. Fraction S-1, obtained through deoxycholate-KCI fractionation procedure, contained one Cyt ofc type (formally cl with Em7.0 of +240 millivolts), two b type Cyt with Em7.. values of +100 and -25 millivolts, ferredoxin-like centers presumably linked to succinic-and NADH-dehydrogenases, and a Rieske-type iron sulfur center (gy = 1.89). The ubiquinol-dependent Cyt c reduction by fraction S-I showed sensitivity to antimycin A, myxothiazol, and n-2-hepthyl-1-hydroxyquinoline N-oxide with I50 of 12 nanomolar, 30 nanomolar, and 0.1 micromblar, respectively. Oxidation-induced extra b type reduction, a widespread phenomenon of bacterial and mitochondrial respiratory systems, has also been observed in both intact mitochondria and S-1 fraction. The data seem to blur previous experiments in which both spectral and functional differences between higher plant and mammalian mitochondria have been underlined.The respiratory chains of eukaryotic and prokaryotic systems are an assembly of more than twenty discrete carriers ofelectrons together with an unspecific number of structural peptides (30). While the function of the respiratory chain as an oxidationreduction driven proton-pump is now accepted (30), the structural basis underlying this function is only partially understood (30). Owing to this, respiratory and photosynthetic electron transport in mammalian mitochondria, photosynthetic bacteria, blue-green algae, and chloroplasts have been analyzed throtgh isolation and characterization of multiple redox complexes (12). However, little effort has been made to try to understand both structural and redox properties of respiratory complexes from plant mitochondria. Published experimentation dealt with partial purification of either the b type Cyt from mung bean (Phaseolus aureus) and potato tuber mitochondria (8,13) (14). Evidence for the presence of a Rieske-type iron-sulfur center (gy = 1.89) is also presented.

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Mucidin and strobilurin A are identical and inhibit electron transfer in the cytochrome bc1 complex of the mitochondrial respiratory chain at the same site as myxothiazol

Cited by 76 publications

References 32 publications

A chemical genomics screen highlights the essential role of mitochondria in HIF-1 regulation

A chemical genomics screen highlights the essential role of mitochondria in HIF-1 regulation

The Mitochondrial Respiratory Chain Is Partially Organized in a Supercomplex Assembly

Functional Characterization and Partial Purification of the Ubiquinol-Cytochrome c Oxidoreductase from Higher Plant Mitochondria (Helianthus tuberosus)

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