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Studies on inhibition of respiratory cytochrome bc1 complex by the fungicide pyrimorph suggest a novel inhibitory mechanism.

Xiao YM, Esser L, Zhou F, Li C, Zhou YH, Yu CA, Qin ZH, Xia D - PLoS ONE (2014)

Bottom Line: All cyt bc1 inhibitors act on either the ubiquinol oxidation (QP) or ubiquinone reduction (QN) site.Using an array of biochemical and spectral methods, we show that pyrimorph acts on an area near the QP site and falls into the category of a mixed-type, noncompetitive inhibitor with respect to the substrate ubiquinol.In silico molecular docking of pyrimorph to cyt b from mammalian and bacterial sources also suggests that pyrimorph binds in the vicinity of the quinol oxidation site.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Chemistry, China Agricultural University, Beijing, China; Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, United States of America.

ABSTRACT
The respiratory chain cytochrome bc1 complex (cyt bc1) is a major target of numerous antibiotics and fungicides. All cyt bc1 inhibitors act on either the ubiquinol oxidation (QP) or ubiquinone reduction (QN) site. The primary cause of resistance to bc1 inhibitors is target site mutations, creating a need for novel agents that act on alternative sites within the cyt bc1 to overcome resistance. Pyrimorph, a synthetic fungicide, inhibits the growth of a broad range of plant pathogenic fungi, though little is known concerning its mechanism of action. In this study, using isolated mitochondria from pathogenic fungus Phytophthora capsici, we show that pyrimorph blocks mitochondrial electron transport by affecting the function of cyt bc1. Indeed, pyrimorph inhibits the activities of both purified 11-subunit mitochondrial and 4-subunit bacterial bc1 with IC50 values of 85.0 μM and 69.2 μM, respectively, indicating that it targets the essential subunits of cyt bc1 complexes. Using an array of biochemical and spectral methods, we show that pyrimorph acts on an area near the QP site and falls into the category of a mixed-type, noncompetitive inhibitor with respect to the substrate ubiquinol. In silico molecular docking of pyrimorph to cyt b from mammalian and bacterial sources also suggests that pyrimorph binds in the vicinity of the quinol oxidation site.

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Difference spectra of inhibitors and inhibitors combinations to reduced Btbc1.All spectra were recorded with purified Btbc1 at a concentration of 5 μM of cyt b with the concentrations of inhibitor as indicated. Prior to spectral scan, the bc1 complex was reduced by addition of dithionite. (A) Spectrum of reduced Btbc1 in the presence of 1 mM pyrimorph (pyr) minus that of reduced Btbc1 alone. (B) Spectrum of reduced Btbc1 in the presence of 10 μM myxothiazol (myx) minus that of reduced Btbc1 alone. (C) and (D) The spectrum of reduced Btbc1 in equilibration with 1 mM pyrimorph followed by addition of 10 μM myxothiazol minus spectrum of reduced Btbc1 in the presence of 10 μM myxothiazol or 1 mM pyrimorph, respectively. (E) Spectrum of reduced Btbc1 in the presence of 10 μM antimycin A (ant) minus spectrum of reduced Btbc1 alone. (F) Spectrum of reduced Btbc1 after equilibration with 1 mM pyrimorph and 10 μM antimycin A in sequence minus spectrum of reduced Btbc1 in the presence of antimycin A.
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pone-0093765-g003: Difference spectra of inhibitors and inhibitors combinations to reduced Btbc1.All spectra were recorded with purified Btbc1 at a concentration of 5 μM of cyt b with the concentrations of inhibitor as indicated. Prior to spectral scan, the bc1 complex was reduced by addition of dithionite. (A) Spectrum of reduced Btbc1 in the presence of 1 mM pyrimorph (pyr) minus that of reduced Btbc1 alone. (B) Spectrum of reduced Btbc1 in the presence of 10 μM myxothiazol (myx) minus that of reduced Btbc1 alone. (C) and (D) The spectrum of reduced Btbc1 in equilibration with 1 mM pyrimorph followed by addition of 10 μM myxothiazol minus spectrum of reduced Btbc1 in the presence of 10 μM myxothiazol or 1 mM pyrimorph, respectively. (E) Spectrum of reduced Btbc1 in the presence of 10 μM antimycin A (ant) minus spectrum of reduced Btbc1 alone. (F) Spectrum of reduced Btbc1 after equilibration with 1 mM pyrimorph and 10 μM antimycin A in sequence minus spectrum of reduced Btbc1 in the presence of antimycin A.

Mentions: Binding of pyrimorph causes the spectrum to red shift, as the difference spectrum [(bc1+pyr)-bc1] shows a trough centered around 565 nm (Fig. 3A), which is an indication that binding of pyrimorph affects cyt b hemes. This spectrum was compared to spectra with bound QP site inhibitor myxothiazol [(bc1+myx)-bc1] and QN site inhibitor antimycin A [(bc1+ant)-bc1], respectively (Figs. 3B and 3E). At a first glance, it seems that the spectral change due to pyrimorph binding resembles that caused by myxothiazol binding, despite considerable differences (see below), indicating that pyrimorph binds closer to the bL heme or the QP site. Indeed, binding pyrimorph to bc1 does not seem to interfere with subsequent binding of antimycin A, as the difference spectrum of [(bc1+pyr+ant) – (bc1+ant)] (Fig. 3F) looks almost identical to [(bc1+pyr)-(bc1)] (Fig. 3A). This experiment confirms that pyrimorph does not target the QN site.


Studies on inhibition of respiratory cytochrome bc1 complex by the fungicide pyrimorph suggest a novel inhibitory mechanism.

Xiao YM, Esser L, Zhou F, Li C, Zhou YH, Yu CA, Qin ZH, Xia D - PLoS ONE (2014)

Difference spectra of inhibitors and inhibitors combinations to reduced Btbc1.All spectra were recorded with purified Btbc1 at a concentration of 5 μM of cyt b with the concentrations of inhibitor as indicated. Prior to spectral scan, the bc1 complex was reduced by addition of dithionite. (A) Spectrum of reduced Btbc1 in the presence of 1 mM pyrimorph (pyr) minus that of reduced Btbc1 alone. (B) Spectrum of reduced Btbc1 in the presence of 10 μM myxothiazol (myx) minus that of reduced Btbc1 alone. (C) and (D) The spectrum of reduced Btbc1 in equilibration with 1 mM pyrimorph followed by addition of 10 μM myxothiazol minus spectrum of reduced Btbc1 in the presence of 10 μM myxothiazol or 1 mM pyrimorph, respectively. (E) Spectrum of reduced Btbc1 in the presence of 10 μM antimycin A (ant) minus spectrum of reduced Btbc1 alone. (F) Spectrum of reduced Btbc1 after equilibration with 1 mM pyrimorph and 10 μM antimycin A in sequence minus spectrum of reduced Btbc1 in the presence of antimycin A.
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Related In: Results  -  Collection

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pone-0093765-g003: Difference spectra of inhibitors and inhibitors combinations to reduced Btbc1.All spectra were recorded with purified Btbc1 at a concentration of 5 μM of cyt b with the concentrations of inhibitor as indicated. Prior to spectral scan, the bc1 complex was reduced by addition of dithionite. (A) Spectrum of reduced Btbc1 in the presence of 1 mM pyrimorph (pyr) minus that of reduced Btbc1 alone. (B) Spectrum of reduced Btbc1 in the presence of 10 μM myxothiazol (myx) minus that of reduced Btbc1 alone. (C) and (D) The spectrum of reduced Btbc1 in equilibration with 1 mM pyrimorph followed by addition of 10 μM myxothiazol minus spectrum of reduced Btbc1 in the presence of 10 μM myxothiazol or 1 mM pyrimorph, respectively. (E) Spectrum of reduced Btbc1 in the presence of 10 μM antimycin A (ant) minus spectrum of reduced Btbc1 alone. (F) Spectrum of reduced Btbc1 after equilibration with 1 mM pyrimorph and 10 μM antimycin A in sequence minus spectrum of reduced Btbc1 in the presence of antimycin A.
Mentions: Binding of pyrimorph causes the spectrum to red shift, as the difference spectrum [(bc1+pyr)-bc1] shows a trough centered around 565 nm (Fig. 3A), which is an indication that binding of pyrimorph affects cyt b hemes. This spectrum was compared to spectra with bound QP site inhibitor myxothiazol [(bc1+myx)-bc1] and QN site inhibitor antimycin A [(bc1+ant)-bc1], respectively (Figs. 3B and 3E). At a first glance, it seems that the spectral change due to pyrimorph binding resembles that caused by myxothiazol binding, despite considerable differences (see below), indicating that pyrimorph binds closer to the bL heme or the QP site. Indeed, binding pyrimorph to bc1 does not seem to interfere with subsequent binding of antimycin A, as the difference spectrum of [(bc1+pyr+ant) – (bc1+ant)] (Fig. 3F) looks almost identical to [(bc1+pyr)-(bc1)] (Fig. 3A). This experiment confirms that pyrimorph does not target the QN site.

Bottom Line: All cyt bc1 inhibitors act on either the ubiquinol oxidation (QP) or ubiquinone reduction (QN) site.Using an array of biochemical and spectral methods, we show that pyrimorph acts on an area near the QP site and falls into the category of a mixed-type, noncompetitive inhibitor with respect to the substrate ubiquinol.In silico molecular docking of pyrimorph to cyt b from mammalian and bacterial sources also suggests that pyrimorph binds in the vicinity of the quinol oxidation site.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Chemistry, China Agricultural University, Beijing, China; Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, United States of America.

ABSTRACT
The respiratory chain cytochrome bc1 complex (cyt bc1) is a major target of numerous antibiotics and fungicides. All cyt bc1 inhibitors act on either the ubiquinol oxidation (QP) or ubiquinone reduction (QN) site. The primary cause of resistance to bc1 inhibitors is target site mutations, creating a need for novel agents that act on alternative sites within the cyt bc1 to overcome resistance. Pyrimorph, a synthetic fungicide, inhibits the growth of a broad range of plant pathogenic fungi, though little is known concerning its mechanism of action. In this study, using isolated mitochondria from pathogenic fungus Phytophthora capsici, we show that pyrimorph blocks mitochondrial electron transport by affecting the function of cyt bc1. Indeed, pyrimorph inhibits the activities of both purified 11-subunit mitochondrial and 4-subunit bacterial bc1 with IC50 values of 85.0 μM and 69.2 μM, respectively, indicating that it targets the essential subunits of cyt bc1 complexes. Using an array of biochemical and spectral methods, we show that pyrimorph acts on an area near the QP site and falls into the category of a mixed-type, noncompetitive inhibitor with respect to the substrate ubiquinol. In silico molecular docking of pyrimorph to cyt b from mammalian and bacterial sources also suggests that pyrimorph binds in the vicinity of the quinol oxidation site.

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