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.
Citation: Xiao Y-M, Esser L, Zhou F, Li C, Zhou Y-H, Yu C-A, et al. (2014) Studies on Inhibition of Respiratory Cytochrome bc1 Complex by the Fungicide Pyrimorph Suggest a Novel Inhibitory Mechanism. PLoS ONE 9(4): e93765. https://doi.org/10.1371/journal.pone.0093765
Editor: Lijun Rong, University of Illinois at Chicago, United States of America
Received: February 20, 2014; Accepted: March 5, 2014; Published: April 3, 2014
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research, and by a grant from the National Basic Research Science Foundation of China (2010CB126100) to ZQ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have the following interests. Dimethomorph was a gift from Jiangshu Frey Chemical Co. Ltd. (Jiangshu Province, China). This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.
The cytochrome bc1 complex (cyt bc1, also known as ubiquinone:cyt c oxidoreductase, Complex III or bc1) is a central component of the cellular respiratory chain of mitochondria. It catalyzes the reaction of electron transfer (ET) from ubiquinol to cyt c and couples this reaction to proton translocation across the mitochondrial inner membrane, contributing to the cross-membrane proton motive force essential for cellular functions such as ATP synthesis , . The indispensible function of cyt bc1 in cellular energy metabolism makes it a prime target for numerous natural and synthetic antibiotics. More than 20 synthetic fungicides targeting cyt bc1 are in widespread use in agriculture with an annual sale exceeding $2.7 billion .
All cyt bc1 inhibitors target either the ubiquinol oxidation site (QP or Qo) or the ubiquinone reduction site (QN or Qi), which are defined by the Q-cycle mechanism of cyt bc1 function , . Despite variations in subunit compositions of bc1 from various organisms, only three subunits are essential for ET-coupled proton translocation function: they are cyt b, cyt c1 and the iron-sulfur protein (ISP). The cyt b subunit contains two b-type hemes (bL and bH), the cyt c1 subunit has a c-type heme, and the ISP possesses a 2Fe-2S cluster. Both active sites are located within the cyt b subunit, as demonstrated by crystallographic studies of mitochondrial and bacterial bc1 complexes –. Resistance to known cyt bc1 fungicides has been reported at an alarming rate, rendering many of these reagents ineffective. Most common mechanisms of resistance involve target site mutations and corresponding strategies to overcome drug resistance have been proposed . Developing new agents targeting areas outside the QP and QN sites of cyt bc1 is most attractive primarily because the new compounds presumably are able to circumvent existing fungal resistance.
Pyrimorph, (Z)-3-[(2-chloropyridine-4-yl)-3-(4-tert-butylphenyl)-acryloyl] morpholine, is a novel systemic antifungal agent that belongs to the family of carboxylic acid amide (CAA) fungicides , whose members include mandipropamid, dimethomorph, flumorph, and valinine derivatives. Pyrimorph exhibits excellent activity inhibiting mycelial growth of the fungal species Phytophthora infestans, Phytophthora capsici, and Rhizoctonia solani and is able to suppress zoosporangia germination of Pseudoperonospora cubensis with EC50 values in the range between 1.3 and 13.5 μM . The in vitro sensitivities of various asexual stages of Peronophythora litchii to pyrimorph were studied with four single-sporangium isolates, showing high sensitivity at the stage of mycelial growth with an EC50 of 0.3 μM .
Although pyrimorph is currently in use to control various fungal pathogens –, its functional mechanism has remained unclear. The presence of a common CAA moiety has led to the suggestion that pyrimorph may work in a fashion similar to that of other CAA-type fungicides . One CAA member, mandipropamid, was shown to target the pathway of cell wall synthesis by inhibiting the CesA3 cellulose synthases . However, treatment of fungal pathogens with pyrimorph appeared to affect multiple cellular pathways, including, but not limited to, those of cellular energy metabolism and cell wall biosynthesis, either directly or indirectly . Indeed, a recent report has correlated the pyrimorph resistance phenotype in P. capsici with mutations in the CesA3 gene .
Other mechanisms of pyrimorph action have yet to be investigated. In particular, its potential interference with cellular respiratory chain components leading to reduced ATP synthesis appears to be a reasonable hypothesis for the observed inhibitory effects on energy demanding processes such as mycelial growth and cytospore germination of fungi. Here, we report the effects of pyrimorph on electron flow through the isolated fungal mitochondrial respiratory chain and the identification of the cyt bc1 complex as pyrimorph’s primary target. Kinetic experiments suggest that the mode of pyrimorph inhibition is to interfere with substrate access to the ubiquinol oxidation site but in a way that differs from other bc1 inhibitors, suggesting a novel mode of inhibitory mechanism.
Materials and Methods
The pyrimorph used in all experiments was synthesized in our laboratory. Dimethomorph was a gift from Jiangshu Frey Chemical Co. Ltd. (Jiangshu Province, China). Cyt c (from horse heart, type III) was purchased from Sigma-Aldrich (St. Louis, MI). 2,3-dimethoxy-5-methyl-6-(10-bromodecyl)-1,4-benzoquinol (Q0C10BrH2) was prepared as previously reported . N-dodecyl-β-D-maltoside (β-DDM) and N-octyl-β-D-glucoside (β-OG) were purchased from Affymetrix (Santa Clara, CA). All other chemicals were purchased and are of the highest grade possible.
Preparation of Light Mitochondria from Phytophthora capsici
Light mitochondrial fraction were prepared from cultured mycelia from laboratory strain Phytophthora capsici Leonia (P. capsici), which was grown in CA liquid medium (8% carrot juice and 2% glucose) for 5 days in the dark at 25°C . 10 g mycelia (fresh weight) were washed with 0.6 M mannitol solution and ground up for 5 minutes with an ice-cold mortar and pestle in 100 ml buffer A containing 10 mM MOPS•KOH, pH 7.1, 0.3 M mannitol, 1 mM EDTA and 0.1% (w/v) bovine serum albumin (BSA) and 30 g of sea sand. The homogenate was centrifuged at 3,200×g for 10 min at 4°C and the supernatant was further centrifuged at 12,000×g for 30 min. The precipitate, light mitochondrial fraction, was resuspended and washed with 20 ml buffer B containing 10 mM MOPS•KOH, pH 7.1, 0.25 M sucrose and 1 mM EDTA and pelleted again by centrifugation at 12,000×g for 20 min at 4°C. The mitochondrial preparation was resuspended in buffer A and the protein concentration was adjusted to 0.1 mg/ml.
Inhibition of the ET Activity of P. capsici Mitochondria by Pyrimorph
The activities of mitochondrial respiratory chain components were assayed using the Mitochondria Complex Activity Assay Kit (Genmed Scientifics, Inc. USA, Wilmington, DE) following manufacturer’s instruction. Briefly, Complex I activity was measured by following the oxidation of NADH by monitoring the decrease in absorbance difference between 340 nm and 380 nm. The reaction mixture (1 ml) consisted of 50 mM potassium phosphate buffer, pH 7.6, 0.25 mM NADH and 50 mM decylubiquinone as the electron acceptor. Crude mitochondria (200 μg protein) were added to start the reaction. Complex II activity was estimated as the rate of reduction of ubiquinone to ubiquinol by succinate, which can be followed by the secondary reduction of 2,6-dichlorophenolindophenol (DCPIP) as the ubiquinol forms. The reaction mixture (1 ml) contained 50 mM potassium phosphate buffer, pH 7.6, 20 mM succinate, 1.0 mM EDTA, 0.05 mM DCPIP and 3 mM NaN3, and 50 mM decylubiquinone. Crude mitochondria (65 μg) were added to initiate the reaction and the decrease in absorbance at 600 nm was followed as DCPIP becomes reduced. Complex III activity was assayed by following the increase in absorbance at 550 nm as cyt c becomes reduced using decylubiquinol as an electron donor. Here, the reaction mixture (1 ml) consisted of 50 mM potassium phosphate buffer, pH 7.6, 0.1% BSA, 0.1 mM EDTA, 60 mM oxidized cyt c, and 150 μM decylubiquinol. Crude mitochondria (10 μg protein) were then added to initiate the reaction.
Purification of Cyt bc1 Complexes from Beef Heart and Photosynthetic Bacterium Rhodobacter sphaeroides
Bovine heart mitochondrial bc1 (Btbc1) complex was prepared starting from highly purified succinate-cyt c reductase, as previously reported . The bc1 particles were solubilized by deoxycholate and contaminants were removed by a 15-step ammonium acetate fractionation. The purified bc1 complex was recovered in the oxidized state from the precipitates formed between 18.5% and 33.5% ammonium acetate saturation. The final product was dissolved in 50 mM Tris•HCl buffer, pH 7.8, containing 0.66 M sucrose resulting in a stock solution with a protein concentration of 30 mg/ml, which was stored at −80°C. The concentrations of cyt b and c1 were determined spectroscopically using millimolar extinction coefficients of 28.5 and 17.5 mM−1 cm−1 for cyt b and c1, respectively.
To prepare cyt bc1 complex from the photosynthetic bacterium R. sphaeroides (Rsbc1), R. sphaeroides strain BC17 cells bearing the pRKD418-fbcFBC6HQ plasmid  were grown photosynthetically at 30°C in an enriched Sistrom medium containing 5 mM glutamate and 0.2% casamino acids . The growth was monitored by measuring the OD600 value every 3–5 h. Cells were transferred to a larger batch or harvested when OD600 reached 1.8–2.0. Chromatophore membranes were prepared from BC17 cells as described previously  and stored at a very high concentration in the presence of 20% glycerol at −80°C. To purify the hexahistidine-tagged Rsbc1 complex, the freshly prepared chromatophores or frozen chromatophores thawed on ice were adjusted to a cyt b concentration of 25 μM with a solubilization buffer containing 50 mM Tris•HCl, pH 8.0 at 4°C, and 1 mM MgSO4. 10% (w/v) β-DDM was added to the chromatophore suspension to a final concentration of 0.56 mg detergent/nmole of cyt b followed by addition of 4M NaCl solution to a final concentration of 0.1 M. After stirring on ice for 1 hour, the admixture was centrifuged at 220,000×g for 90 minutes; the supernatant was collected and diluted with equal volume of the solubilization buffer followed by passing through a Ni-NTA agarose column (100 nmole of cyt b/ml of resin) pre-equilibrated with two volumes of the solubilization buffer. After loading, the column was washed sequentially with the following buffers until the absence of greenish color in effluent was reached: washing buffer (50 mM Tris•HCl, pH 8.0 at 4°C, containing 100 mM NaCl) +0.01% β-DDM; washing buffer +0.01% β-DDM and 5 mM histidine; washing buffer +0.5% β-OG; washing buffer +0.5% β-OG and 5 mM histidine. The cyt bc1 complex was eluted with the washing buffer +0.5% β-OG and 200 mM histidine. Pure fractions were combined and concentrated by Centriprep-30 concentrator. Glycerol was added to a final concentration of 10% before storage at −80°C.
Measurement of bc1 Activity and its Inhibition (IC50) by Various Inhibitors
The activities of isolated cyt bc1 complexes were assayed following the reduction of substrate cyt c. The purified bc1 complexes were diluted to a final concentration of 0.1 μM and 1 μM based on the concentration of cyt b for Btbc1 and Rsbc1, respectively, in the B200 buffer (50mM Tris•HCl, pH 8.0, 0.01% β-DDM, 200 mM NaCl). The assay mixture contains 100 mM phosphate buffer, pH 7.4, 0.3 mM EDTA, and 80 μM cyt c, and Q0C10BrH2 at a final concentration of 5 μM. The addition of 3 μl of diluted bc1 solution initiates the reaction, which is recorded immediately following the cyt c reduction at 550 nm wavelength for 100 seconds in a two-beam Shimadzu UV-2250 PC spectrophotometer at 23°C. The amount of cyt c reduced over a given period of time was calculated using a millimolar extinction coefficient of 18.5 mM−1 cm−1.
To measure the effect of bc1 inhibitors, bc1 was pre-incubated at various concentrations of an inhibitor for 15 minutes prior to the measurement of its activity. The IC50 value was calculated by a least-squares procedure fitting the equation (Y = Amin+(Amax–Amin)/(1+10(X−logIC50)) implemented in the commercial package Prism, where Amax and Amin are maximal and minimal activities, respectively. Although the chemical properties of Q0C10BrH2 are comparable to those of Q0C10H2, the former is a better substrate for the cyt bc1 complex isolated in detergent solution .
Reaction Kinetics of bc1 in the Presence of Inhibitors
To measure the enzyme kinetics of cyt bc1 complex under inhibitory conditions, purified cyt bc1, either Btbc1 or Rsbc1, was assayed at different concentrations of substrates. When the Q0C10BrH2 concentration was varied (1 μM, 2 μM, 5 μM, 10 μM, 20 μM) the cyt c concentration was kept constant at 80 μM, whereas when the concentration of cyt c varied (1 μM, 2 μM, 4 μM, 8 μM, 12 μM, 16 μM) the Q0C10BrH2 concentration was kept constant at 50 μM. The reactions were initiated by adding 3 μl of diluted bc1 solution (0.1 μM for Btbc1 or 1.0 μM for Rsbc1) pre-incubated with various concentrations of inhibitors for 15 minutes. The time course of the absorbance change due to cyt c reduction was recorded continuously at 550 nm. Initial rates were determined from the slopes in the linear portion of cyt c1 reduction time course.
Analysis of Cyt bc1 Spectra in the Presence of Inhibitors
For each run a solution of 1 ml bovine cyt bc1 at a cyt b concentration of 5 μM was fully reduced with addition of a tiny amount of sodium dithionite and its spectrum was obtained in the range of 520–600 nm. A specific inhibitor was added at various concentrations to the reduced bc1 complex and was scanned repeatedly until no changes were observed. All scans were stored digitally and difference spectra were produced by subtracting the corresponding spectrum of the inhibitor-free, fully reduced bc1 complex.
Measurement of Cyt b and c1 Reduction Time Course in a Single Turnover Reaction
The enzyme was diluted to a final concentration of about 4 μM of cyt c1 in 1 ml of B200 buffer (50 mM Tris•HCl, pH 8.0, 0.01% β-DDM, 200 mM NaCl) and oxidized fully by adding a tiny amount of potassium ferricyanide. The spectrum of the fully oxidized enzyme in the range of 520–590 nm was stored. Inhibitors at various concentrations were introduced and incubated for 2 min followed by addition of the ubiquinol analog Q0C10BrH2 to a final concentration of 10 μM to start the reaction. Spectra were recorded at 20-second intervals starting immediately after mixing. After 800 seconds the enzyme was fully reduced by dithionite. The spectrum of the fully oxidized complex was subtracted from that at each time point and the amounts of reduced cyt c1 and b at a given time were calculated from the difference spectra at 552–540 nm and 560–576 nm, respectively.
Molecular Docking of Pyrimorph to Cyt b
Coordinates for the receptor molecule were taken from the protein data bank (pdb) entry 1SQX, for the stigmatellin inhibited complex of Btbc1. The side chains of residues E271 and F274 were modeled as standard rotamers consistent with their positions in apo Btbc1. The ligand molecule, pyrimorph, was drawn and converted to a SMILES string using tools from the CADD Group . The SMILES string was converted by the program Elbow  to 3D coordinates and energy minimized in GAMESS . Both receptor and ligand molecules were converted to standard pdbqt files using mgltools . For all docking runs, the acrylamide moiety (C7 = C8–C9 = O16) of pyrimorph was fixed in the syn-periplanar conformation because the alternative anti- conformation would bring the larger morpholino and pyridyl groups into close contact.
Two approaches to docking were taken: (1) as the most likely binding sites for inhibitors are the QP and the QN sites, docking attempts were made first at those known sites. (2) In a second set of runs, no prior knowledge of sites was imposed but the program Q-site-finder  was used to locate all potential binding sites and docking was carried out at all sensible locations. Docking pyrimorph to known and unknown inhibitor binding sites was performed using the program Autodock Vina .
Pyrimorph Blocks the Mitochondrial Respiratory Chain by Targeting cyt bc1 Complex
To test whether pyrimorph inhibits fungal growth by interfering with the cellular energy metabolism pathway, in particular the mitochondrial respiratory chain, we isolated light mitochondrial fraction from the pathogenic fungus P. capsici and examined the ability of pyrimorph to inhibit various segments of the respiratory chain (Table 1). It is quite clear that pyrimorph has no effect on the activity of Complex I, as a concentration of pyrimorph as high as 16 μM was unable to inhibit NADH oxidation catalyzed by Complex I. By contrast, under the same conditions, pyrimorph inhibits 94.6% of Complex II activity. Most importantly, Complex III, the cyt bc1 complex, shows the highest sensitivity toward pyrimorph, with 95.3% inhibition even at 4 μM concentration.
Although the effects of pyrimorph on the mitochondrial respiratory chain of P. capsici were clearly demonstrated, such effects could be indirect. To ascertain that the target of pyrimorph is indeed the cyt bc1 complex, we used highly purified cyt bc1 from beef heart (Bos taurus bc1 or Btbc1) and assayed inhibition of its cyt c reductase activity by pyrimorph. The result was compared to the well-known anti-bc1 fungicide azoxystrobin and two other CAA-type fungicides, dimethomorph and flumorph. As shown in Fig. 1A, Btbc1 activities are reduced to 67% and 42% of the control, respectively, in the presence of 10 μM and 100 μM of pyrimorph. Azoxystrobin is able to inhibit bc1 activity by more than 95% at 10 μM concentration. However, the two CAA-type fungicides, dimethomorph and flumorph, displayed no activity at all against Btbc1. These results indicated that pyrimorph is different from other members of CAA-type fungicides and more importantly established that pyrimorph is indeed an inhibitor of the cyt bc1 complex, albeit a weak one.
(A) Inhibition of Btbc1 by several amide fungicides and azoxystrobin at indicated concentrations. The control is the activity of bc1 in the absence of inhibitor, which is set to 100%. (B) Concentration-dependent inhibition of Btbc1 by pyrimorph. (C) Concentration-dependent inhibition of Rsbc1 by pyrimorph.
We subsequently determined 50% inhibitory concentration (IC50) for pyrimorph against isolated cyt bc1 complexes from both bovine mitochondria and photosynthetic bacterium R. sphaeroides (Rsbc1). Pyrimorph is slightly more potent against Rsbc1, giving an IC50 value of 69.2 μM; it gives an IC50 of 85.0 μM for Btbc1 (Figs. 1B and 1C). As a comparison, the well-known bc1 inhibitor stigmatellin and azoxystrobin give IC50 values of 2.8 nM and 47.7 nM, respectively, for isolated Btbc1 by our measurement (data not shown) under the same assay conditions. Since Rsbc1 has only four subunits, it is therefore certain that pyrimorph targets the essential subunits of the bc1 complex.
Effect of Pyrimorph Binding on Reduction of the Cyt b and c1 by Ubiquinol
Nearly all cyt bc1 inhibitors bind to the QN site, QP site or both . It is known that binding of inhibitors produces various effects on spectra of cyt b and c1 heme groups, as well as on redox potential and conformation of the iron-sulfur protein –. These effects ultimately determine the rate and amount of cyt b or c1 reduced under equilibrium conditions and can be exploited to compare modes of action of different inhibitors , distinguishing for example a QN site inhibitor from a QP site one. Starting with a completely oxidized enzyme, Btbc1 was mixed with substrate Q0C10BrH2 in the presence of pyrimorph at two different concentrations (0.1 mM and 1.0 mM); no cyt c was used as the terminal electron acceptor. The amount of cyt b including bL and bH hemes and cyt c1 reduced as a function of time was recorded (Figs. 2A and 2B). The results were compared with those produced by QN site inhibitor antimycin A (Fig. 2C) and by QP site inhibitor myxothiazol (Fig. 2D).
Isolated Btbc1 was incubated with indicated inhibitors followed by single turn-over reaction initiated by addition of 10 μM Q0C10BrH2. The spectra were recorded immediately following the mixing and every 20 seconds thereafter. At the 800 second time point, a tiny amount of sodium dithionite was added to reduce both cyt b and c1. The amounts of reduced cyt b and c1 were calculated and plotted as a function of time. The green trace is the amount of cyt b reduced over time and the red one is the amount of cyt c1 reduced. (A) 100 μM pyrimorph, (B), 1000 μM pyrimorph, (C) 30 μM myxothiazol, and (D) 30 μM antimycin A.
In the absence of the high-potential electron acceptor cyt c, only a single enzymatic turnover at the QP site is possible when the QN site inhibitor antimycin A is bound. Under such conditions, both cyt b and c1 were rapidly reduced reaching maximal reduction of nearly half of the b-type hemes and all of the c-type heme perhaps even before the first measurement was recorded (Fig. 2C). Once reaching maximal reduction, cyt b began non-enzymatic oxidation rather rapidly, whereas the redox state of cyt c1 remained unchanged. When the QP site is occupied by an inhibitor such as myxothiazol, not a single turnover is possible (Fig. 2D). The initial rapid reduction of cyt b was most likely via the revere reaction at the QN site and cyt c1 reduction was entirely non-enzymatic. Thus, the rate of cyt c1 reduction and that of cyt b re-oxidation can be employed to determine which site an inhibitor targets.
Clearly, the reduction behavior of cytochromes b and c1 induced by pyrimorph distinguishes it from that induced by antimycin A (Figs. 2A, 2B and 2C), demonstrating that pyrimorph does not target the QN site. By contrast, the time courses of cyt b and c1 reduction in the presence of pyrimorph or myxothiazol resemble each other (Figs. 2A, 2B, and 2D), except that the latter inhibitor takes a longer time for cyt c1 to be maximally reduced than pyrimorph does. This result puts pyrimorph into the category of a QP-site inhibitor by either directly or indirectly competing with ubiquinol for the QP site.
Spectral Analyses Suggest the Binding of Pyrimorph near QP Site
Single turnover experiments suggested that pyrimorph acts in a fashion similar to that of QP site inhibitors. It was thus necessary to determine how pyrimorph interferes with bc1 function at the QP site. Since spectral changes, especially red shifts in the α- and β-band caused by binding of inhibitors to reduced bc1, were successfully used to deduce information about their binding interactions , , a similar approach was taken for pyrimorph. By comparing the spectra of reduced bc1 bound with pyrimorph to those obtained with known QP or QN site inhibitors such as myxothiazol or antimycin A, we hoped to gain insight into the binding interactions and location.
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.
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.
However, binding of pyrimorph to bc1 does affect subsequent binding of myxothiazol and vise versa, because the difference spectrum [(bc1+pyr+myx) – (bc1+myx)] (Fig. 3C) does not look like that of [(bc1+pyr)-(bc1)] (Fig. 3A), nor does the difference spectrum [(bc1+pyr+myx) – (bc1+pyr)] (Fig. 3D) resemble that of [(bc1+myx)-(bc1)] (Fig. 3B). These spectra indicate the possibility that both inhibitors can co-exist near the QP pocket and influence each other. Since the binding of myxothiazol to the QP pocket is well established, the experiment further suggests that pyrimorph may have a different binding mode from that of myxothiazol.
Inhibitory Kinetics of Cyt bc1 Suggests the Mode of Pyrimorph Action
The possibility that pyrimorph has a different mode of action is of particular interest in light of our extensive knowledge on the development of resistance to existing inhibitors. To further probe the mechanism of pyrimorph’s action, we investigated the kinetic properties of bc1 function under pseudo first-order reaction conditions by measuring its activity with respect to changes in concentration of either substrates Q0C10BrH2 or cyt c in the presence of different amount of pyrimorph, allowing double reciprocal or Lineweaver-Burk plots to reveal the relationship between 1/V and 1/[S]. The measurements were done for both Btbc1 and Rsbc1, revealing nearly identical kinetic behavior (Fig. 4). As shown in Figs. 4A and 4C, in the presence of a constant 80 μM cyt c and with increasing concentrations of Q0C10BrH2, both Km and Vmax are altered as is the Km/Vmax ratio. Expectedly, as the concentration of pyrimorph increases, the Vmax decreases; at a constant pyrimorph concentration, the reciprocal enzyme activity 1/V has a positive slope with respect to 1/[S]. However, the Km value for the substrate quinol changed, falling between competitive (Fig. 4E) and non-competitive (Fig. 4F) inhibitions. Thus, pyrimorph falls into the category of a mixed-type, noncompetitive inhibitor with respect to the substrate ubiquinol, suggesting that it competes, both directly and indirectly, with ubiquinol to occupy the QP site.
Four different concentrations of 0, 10, 100 and 1000 μM were used for pyrimorph and three, 0, 30 and 50 nM were used for myxothiazol. Each point represents a mean value of at least 3 independent experimental measurements. (A) Inhibition of Btbc1 by pyrimorph with variations in concentration of Q0C10BrH2. (B) Inhibition of Btbc1 by pyrimorph with variations in concentration of cyt c. (C) Inhibition of Rsbc1 by pyrimorph with variations in concentration of Q0C10BrH2. (D) Inhibition of Rsbc1 by pyrimorph with variations in concentration of cyt c. (E) Inhibition of Btbc1 by myxothiazol with variations in concentration of Q0C10BrH2. (F) Inhibition of Btbc1 by myxothiazol with variations in concentration of cyt c.
At a constant 50 μM Q0C10BrH2 concentration and with varying concentrations of cyt c, double-reciprocal plots show that x-intercepts remain the same with or without pyrimorph (Figs. 4B and 4D), suggesting that the apparent Km for substrate cyt c remains unchanged. Thus, pyrimorph is a noncompetitive inhibitor with respect to cyt c. As a control, we performed the same experiments with Btbc1 using the classic QP-site inhibitor myxothiazol, showing that myxothiazol is a competitive inhibitor for the substrate quinol but a non-competitive inhibitor for cyt c (Figs. 4E and 4F).
Docking of Pyrimorph to cyt b Subunit
Docking of pyrimorph to known inhibitor-binding sites in the cyt b subunit of Btbc1 were performed with Autodock Vina and resulted in top solutions at the QP site with a binding free energy of −9.7 kcal/mol and −9.2 kcal/mol at the QN site, representing a 2.3-fold difference in binding affinity between the two sites. These energy values can be compared with binding of other known bc1 inhibitors such as stigmatellin, giving rise to a binding free energy of −10.5 kcal/mol. Potential inhibitor binding sites outside the known active sites were searched by Q-site-finder and the top 20 sites suggested (which included the QP and QN site) were subjected to extensive docking trials using Autodock Vina but no new locations showing improved affinity over the classic sites were identified. The QP site showed the highest binding affinity to pyrimorph. Unlike traditional inhibitors, pyrimorph does not enter the QP site, but rather blocks the entrance or portal to the quinol oxidation site (Fig. 5A). While its morpholino and 4-(2-chloro pyridyl) moieties stay in the central cavity of the bc1 dimer, its 4-(tert-butyl) phenyl group enters the access portal, where it is stabilized by the aromatic side chain of F274 and partially by F128. The latter primarily interacts with the pyridyl moiety via aromatic-aromatic (Ar-Ar) interactions. The tert-butyl group that penetrates into the QP site is flanked by the residues Y273, Y131 and P270, establishing beneficial van-der-Waals contacts.
(A) Molecular surface of the cyt b subunit is given, showing the access portal leading to the QP site, which is blocked by the docked pyrimorph in a ball-and-stick model with atoms of carbon in magenta, nitrogen in blue, oxygen in red and chlorine in green. (B) Stereoscopic pair showing the detailed interactions between residues in the cyt b subunit with the docked pyrimorph molecule.
Resistance to cyt bc1 inhibitors has been extensively investigated, revealing a wide variety of underlying mechanisms including target site mutations , , activation of alternative oxidase pathways , , altered metabolic degradation , reduced uptake and increased efflux , . By far, target site mutation is the most prevalent form of resistance that develops against bc1 inhibitors. Thus, extensive research has been focusing on how to overcome resistance caused by target site mutations. Finding inhibitors that target alternative sites seems to be an attractive strategy.
Pyrimorph is a Multi-target Fungicide Displaying Inhibitory Activity against Cyt bc1
Pyrimorph is a fungicide containing a carboxylic acid amide (CAA) moiety and was shown to be cross-resistant with other CAA fungicides such as mandipropamid, dimethomorph and flumorph , suggesting the possibility that pyrimorph may function in a manner similar to that of other CAA-type fungicides such as mandipropamid for which the mode of action was established by inhibiting cellulose synthase 3 or CesA3 . In a recent publication , pyrimorph-resistant isolates of P. capsici were selected in the presence of the inhibitor and the three most resistant strains share a common mutation (Q1077K) in the CesA3 gene, which is different from the one (G1104V) selected for mandipropamid resistance . However, it remains to be seen whether transfer of the resistant allele to the sensitive parental strain would make the latter pyrimorph-resistant. So far there is no direct evidence from in vitro biochemical experiments that shows at the protein level the inhibition of CesA3 by pyrimorph.
In the current study, we followed up on the previous observation that pyrimorph may act on the cellular respiratory chain of pathogenic fungi . We showed that pyrimorph is able to suppress the respiratory chain function at 4 μM concentration by inhibiting the activity of Complex III in isolated mitochondria of P. capsici mycelia (Table 1). We further showed conclusively that pyrimorph inhibits purified mitochondrial as well as bacterial bc1 complexes with IC50 values at sub-millimolar range (Fig. 1). By contrast, two other CAA-type inhibitors, dimethomorph and flumorph, displayed no inhibitory activity against bc1 complex (Fig. 1A).
It did not escape our notice that cyt bc1 in light mitochondrial fraction isolated from P. capsici mycelia appears to be more sensitive to pyrimorph than purified bovine or bacterial bc1 complexes, suggesting the following possibilities: (1) Direct comparison between results of two very different assays is not a fair comparison, because in isolated light mitochondrial fraction the estimation of cyt bc1 concentration is difficult in the presence of many different proteins. However, the inhibitory concentrations or IC values are directly related to the amount of enzyme in the assay solution. So the lower IC value could be due to a lower concentration of bc1 in the assay conditions. (2) Being a hydrophobic compound, pyrimorph may preferentially partition into the lipid bilayer of mitochondrial membranes, leading to a higher local concentration and in turn to the apparent 95% inhibition at 4 μM concentration (Table 1, Fig. 1). (3) Conversely, the presence of detergent (micelles) in the solution of purified bc1 complex might lower the effective concentration of pyrimorph. This scenario is less likely, as the concentration of β-DDM in our assay buffer is barely above one critical micelle concentration (CMC). (4) The cyt bc1 of P. capcisi is more sensitive to pyrimorph than either bovine or bacterial bc1. However, we note that the purified bacterial complex is more sensitive to pyrimorph than bovine bc1 but only by a factor of 1.2. The difference might simply be a reflection of changes in the sequences and we do observe that bacterial bc1 exhibits slightly higher similarity to fungal than bovine mitochondrial cyt b.
Pyrimorph Likely Acts Near but not at the QP Site
The fact that pyrimorph inhibits both 11-subunit Btbc1 and 4-subunit Rsbc1 demonstrates that the inhibitor acts on cyt b, cyt c1 or ISP subunits of the complex; it does not inhibit mitochondrial bc1 function through binding to the so-called supernumerary subunits (Figs. 1B and 1C). The two potential sites for pyrimorph binding are QN and QP in the cyt b subunit and so far all experimental evidence suggests a binding site near the QP site: (1) Single turn-over experiments show the reduction rate of cyt c1 and re-oxidation rate of cyt b in the presence of two different concentrations of pyrimorph (Figs. 2A and 2B) are very similar to those in the presence of the QP site inhibitor myxothiazol (Fig. 2D), but are drastically different from those in the presence of the QN site inhibitor antimycin A (Fig. 2C). (2) Difference spectra of reduced cyt bc1 also provided strong evidence that pyrimorph targets the QP site (Fig. 3), because the difference spectrum of [(bc1+pyr)-(bc1)] (Fig. 3A) resembles that of [(bc1+myx)-(bc1)] (Fig. 3B) but not that of [(bc1+ant)-(bc1)] (Fig. 3E).
While the analysis of the difference spectra points to the QP pocket as the target site, it also suggests that pyrimorph has a non-overlapping binding site with the classic QP site inhibitor myxothiazol (Fig. 3). Indeed, double-reciprocal or Lineweaver-Burk plots of bc1 activity showed that pyrimorph acts as a mixed-type, non-competitive inhibitor with respect to the substrate ubiquinol (Figs. 4A and 4C), suggesting that pyrimorph may act both competitively and non-competitively for the substrate ubiquinol (Fig. 4E). Mechanistically, it means that pyrimorph is capable of modulating the binding of the substrate ubiquinol without directly competing with it at the active site, which categorizes it as a mixed-type, non-competitive inhibitor .
Unlike classic QP site inhibitors that compete directly with substrate ubiquinol for interactions with the same set of residues in the QP site, pyrimorph rather seems to block the portal to the QP site, through which the substrate ubiquinol has to pass to contact ISP. Simultaneously, a good portion of pyrimorph is held outside the substrate-binding pocket by hydrophobic forces. Consequently, ubiquinol has to actively displace pyrimorph from the entrance in order to gain access to the QP site as in the case of a competitive inhibitor. On the other hand, as pyrimorph has the ability to adhere well to the lipophilic sides of the portal that leads to the QP site of cyt b, it may stay close and possibly interfere with the necessary motion of the cd1/cd2 helix and with the release of ubiquinone, displaying inhibitory activities more characteristic of non-competitive inhibitors. This picture is entirely consistent with biochemical and spectral characterizations of pyrimorph, qualifying it as a mixed-type, non-competitive inhibitor.
Molecular modeling of other CAA-type fungicides such as dimethomorph indicate a very similar binding position and orientation to the QP site but with a significantly lower free energy for binding, consistent with the observation that both dimethomorph and flumorph are not bc1 inhibitors (Fig. 1A). Since both dimethomorph and flumorph structurally resemble pyrimorph, it is clear that the shape of pyrimorph is not a dominant factor for its ability to bind bc1. At the very least the binding of pyrimorph has sparked ideas for a dual approach to inhibitor design: (1) on the side that binds to the QP entrance, modifications could significantly increase the affinity, making it a better competitive inhibitor. (2) Improvements in hydrophobicity or geometric factors on the side that stays outside the QP pocket, the inhibitor could enhance its non-competitive properties. Should it be possible to design a dual type inhibitor, fungal resistance may be stalled for an extended period. Suggestions for improvements might include modifications of the tert-butyl group to include polar groups (hydroxy methyl, methoxy methyl, etc.) that are within the reach of E271 (both sidechain and backbone amide) as well as sterically demanding aromatic groups that may take advantage of the large, aromatic cavity of the QP site. On the side that stays outside of the QP pocket, variations of saturated and aromatic ring systems seem likely to improve binding properties, as it appears that the morpholino group and the chloro-pyridyl group can change places with minimal change in binding energy.
We thank George Leiman for editorial assistance during the preparation of this manuscript. This study utilized the high-performance computational capabilities of the Biowulf Linux cluster at the National Institutes of Health, Bethesda, MD (http://biowulf.nih.gov).
Conceived and designed the experiments: YMX ZHQ DX. Performed the experiments: YMX LE FZ CL YHZ DX. Analyzed the data: YMX LE DX. Contributed reagents/materials/analysis tools: CAY. Wrote the paper: YMX LE DX.
- 1. Trumpower BL (1990) Cytochrome bc1 complexes of microorganisms. Microbiological Reviews 54: 101–129.
- 2. Keilin D (1925) On cytochrome, a respiratory pigment, common to animals, yeast, and higher plants. Proc R Soc Lond B98: 312–399.
- 3. Leadbeater A (2012) Resistance risk to QoI fungicides and anti-resistance strategies. In: Thind TS, editor. Fungicide resistance in crop protection: risk and mangement: CAB eBooks. 141–152.
- 4. Trumpower BL (1990) The protonmotive Q cycle. Energy transduction by coupling of proton translocation to electron transfer by the cytochrome bc1 complex. Journal of Biological Chemistry 265: 11409–11412.
- 5. Mitchell P (1975) Protonmotive redox mechanism of the cytochrome b-c1 complex in the respiratory chain: protonmotive uniquinone cycle. FEBS Lett 56: 1–6.
- 6. Xia D, Yu CA, Kim H, Xia JZ, Kachurin AM, et al. (1997) Crystal structure of the cytochrome bc1 complex from bovine heart mitochondria. Science 277: 60–66.
- 7. Iwata S, Lee JW, Okada K, Lee JK, Iwata M, et al. (1998) Complete structure of the 11-subunit bovine mitochondrial cytochrome bc1 complex [see comments]. Science 281: 64–71.
- 8. Zhang Z, Huang L, Shulmeister VM, Chi YI, Kim KK, et al. (1998) Electron transfer by domain movement in cytochrome bc1. Nature 392: 677–684.
- 9. Hunte C, Koepke J, Lange C, Rossmanith T, Michel H (2000) Structure at 2.3 A resolution of the cytochrome bc(1) complex from the yeast Saccharomyces cerevisiae co-crystallized with an antibody Fv fragment. Structure 15: 669–684.
- 10. Esser L, Elberry M, Zhou F, Yu CA, Yu L, et al. (2008) Inhibitor complexed structures of the cytochrome bc1 from the photosynthetic bacterium Rhodobacter sphaeroides at 2.40 Å resolution. J Biol Chem 283: 2846–2857.
- 11. Kleinschroth T, Castellani M, Trinh CH, Morgner N, Brutschy B, et al. (2011) X-ray structure of the dimeric cytochrome bc(1) complex from the soil bacterium Paracoccus denitrificans at 2.7-A resolution. Biochim Biophys Acta 1807: 1606–1615.
- 12. Berry EA, Huang L, Saechao LK, Pon NG, Valkova-Valchanova M, et al. (2004) X-ray structure of Rhodobacter capsulatus cytochrome bc1: comparison with its mitochondrial and chloroplast counterparts. Photosynthesis Research 81: 251–275.
- 13. Esser L, Yu CA, Xia D (2013) Structural Basis of Resistance to Anti-Cytochrome bc1 Complex Inhibitors: Implication for Drug Improvement. Curr Pharm Des.
- 14. Mu CW, Yuan HZ, Li N, Fu B, Xiao YM, et al. (2007) Synthesis and fungicidal activities of a novel seris of 4-[3-(pyrid-4-yl)-3-substituted phenyl acryloyl] morpholine. Chem J Chinese U 28: 1902–1906.
- 15. Chen XX, Yuan HZ, Qin ZH, Qi SH, Sun LP (2007) Preliminary studies on antifungal activity of pyrimorph. Chinese Journal of Pesticide Science 9: 229–234.
- 16. Wang HC, Sun HY, Stammler G, Ma JX, Zhou MG (2009) Baseline and differential sensitivity of Peronophythora litchii (lychee downy blight) to three carboxylic acid amide fungicides. Plant Pathology.
- 17. Du YN, Wang GZ, Li G (2008) Preventive and therapeutic experiment of 20% Bimalin on phytophthora blight of Capsicum. Modern Agrochemicals 7: 44–46.
- 18. Sun H, Wang H, Stammler G, Ma J, Zhou MG (2010) Baseline Sensitivity of Populations of Phytophthora capsici from China to Three Carboxylic Acid Amide (CAA) Fungicides and Sequence Analysis of Cholinephosphotranferases from a CAA-sensitive Isolate and CAA-resistant Laboratory Mutants. Journal of Phytopathology 158: 244–252.
- 19. Blum M, Boehler M, Randall E, Young V, Csukai M, et al. (2010) Mandipropamid targets the cellulose synthase-like PiCesA3 to inhibit cell wall biosynthesis in the oomycete plant pathogen, Phytophthora infestans. Mol Plant Pathol 11: 227–243.
- 20. Yan X, Qin W, Sun L, Qi S, Yang D, et al. (2010) Study of inhibitory effects and action mechanism of the novel fungicide pyrimorph against Phytophthora capsici. J Agric Food Chem 58: 2720–2725.
- 21. Pang Z, Shao J, Chen L, Lu X, Hu J, et al. (2013) Resistance to the novel fungicide pyrimorph in Phytophthora capsici: risk assessment and detection of point mutations in CesA3 that confer resistance. PLoS One 8: e56513.
- 22. Yu CA, Yu L (1982) Syntheses of biologically active ubiquinone derivatives. Biochemistry 21: 4096–4101.
- 23. Mitani S, Araki S, Takii Y, Ohshima T, Matsuo N, et al. (2001) The biochemical mode of action of the novel selective fungicide cyazofamid: specific inhibition of mitochondrial complex III in Phythium spinosum. Pesticide Biochemistry and Physiology 71: 107–115.
- 24. Yu L, Yang S, Yin Y, Cen X, Zhou F, et al. (2009) Chapter 25 Analysis of electron transfer and superoxide generation in the cytochrome bc1 complex. Methods Enzymol 456: 459–473.
- 25. Mather MW, Yu L, Yu CA (1995) The involvement of threonine 160 of cytochrome b of Rhodobacter sphaeroides cytochrome bc1 complex in quinone binding and interaction with subunit IV. Journal of Biological Chemistry 270: 28668–28675.
- 26. Tian H, Yu L, Mather MW, Yu CA (1997) The Involvement of Serine 175 and Alanine 185 of Cytochrome b of Rhodobacter sphaeroides Cytochrome bc1 Complex in Interaction with Iron-Sulfur Protein. Journal of Biological Chemistry 272: 23722–23728.
- 27. Yu L, Yu CA (1991) Essentiality of the molecular weight 15,000 protein (subunit IV) in the cytochrome b-c1 complex of rhodobacter sphaeroides. Biochemistry 30: 4934–4939.
- 28. Nicklaus M.C SM (2012) CADD Group Chemoinformatics Tools and User Services.
- 29. Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, et al. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallographica Section D 66: 213–221.
- 30. Schmidt MW, Baldridge KK, Boatz JA, Elbert ST, Gordon MS, et al. (1993) General Atomic and Molecular Electronic-Structure System. Journal of Computational Chemistry 14: 1347–1363.
- 31. Sanner MF (1999) Python: A programming language for software integration and development. Journal of Molecular Graphics and Modelling 17: 57–61.
- 32. Laurie AT, Jackson RM (2005) Q-SiteFinder: an energy-based method for the prediction of protein-ligand binding sites. Bioinformatics 21: 1908–1916.
- 33. Trott O, Olson AJ (2010) AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. Journal of Computational Chemistry 31: 455–461.
- 34. Esser L, Quinn B, Li Y, Zhang M, Elberry M, et al. (2004) Crystallographic studies of quinol oxidation site inhibitors: A modified classification of inhibitors for the cytochrome bc1 complex. Journal of Molecular Biology 341: 281–302.
- 35. von Jagow G, Link TA (1986) Use of specific inhibitors on the mitochondrial bc1 complex. Methods in Enzymology 126: 253–271.
- 36. Link TA, Haase U, Brandt U, von Jagow G (1993) What information do inhibitors provide about the structure of the hydroquinone oxidation site of ubihydroquinone: cytochrome c oxidoreductase? Journal of Bioenergetics and Biomembrane 25: 221–232.
- 37. von Jagow G, Liungdahl PO, Craf P, Ohnishi T, Trumpower BL (1984) An Inhibitor of Mitochondrial Respiration Which Binds to Cytochrome b and Displaces Quinone from the Iron-Sulfur Protein of the Cytochrome bc1 Complex. Journal of Biological Chemistry 259: 6318–6326.
- 38. von Jagow G, Engel WD (1981) Complete inhibition of electron transfer from ubiquinol to cytochrome b by teh combined action of antimycin and myxothiazol. FEBS Letters 136: 19–24.
- 39. Berry EA, Huang LS, Lee DW, Daldal F, Nagai K, et al. (2010) Ascochlorin is a novel, specific inhibitor of the mitochondrial cytochrome bc1 complex. Biochim Biophys Acta 1797: 360–370.
- 40. Jordan DB, Livingston RS, Bisaha JJ, Duncan KE, Pember SO, et al. (1999) Mode of action of famoxadone. Pesticide Science 55: 105–118.
- 41. Bolgunas S, Clark DA, Hanna WS, Mauvais PA, Pember SO (2006) Potent inhibitors of the Qi site of the mitochondrial respiration complex III. J Med Chem 49: 4762–4766.
- 42. Brasseur G, Saribas AS, Daldal F (1996) A compilation of mutations located in the cytochrome b subunit of the bacterial and mitochondrial bc1 complex. BiochimBiophysActa 1275: 61–69.
- 43. Wood PM, Hollomon DW (2003) A critical evaluation of the role of alternative oxidase in the performance of strobilurin and related fungicides acting at the Qo site of complex III. Pest Manag Sci 59: 499–511.
- 44. Steinfeld U, Sierotzki H, Parisi S, Poirey S, Gisi U (2001) Sensitivity of mitochondrial respiration to different inhibitors in Venturia inaequalis. Pesticide Mangement Science 57: 787–796.
- 45. Dosnon-Olette R, Schroder P, Bartha B, Aziz A, Couderchet M, et al. (2011) Enzymatic basis for fungicide removal by Elodea canadensis. Environ Sci Pollut Res Int 18: 1015–1021.
- 46. Gaur M, Choudhury D, Prasad R (2005) Complete inventory of ABC proteins in human pathogenic yeast, Candida albicans. J Mol Microbiol Biotechnol 9: 3–15.
- 47. Hill P, Kessl J, Fisher N, Meshnick S, Trumpower BL, et al. (2003) Recapitulation in Saccharomyces cerevisiae of cytochrome b mutations conferring resistance to atovaquone in Pneumocystis jiroveci. Antimicrob Agents Chemother 47: 2725–2731.
- 48. Garrett RH, Grisham CM (1999) Biochemistry. Fort Worth, Philadelphia, San Diego, New York, Orlando, Austin, San Antonio, Toronto, Montreal, London, Sydney, Tokyo: Saunders College Publishing, Harcourt Brace College Publishers.
- 49. Yoshida T, Murai M, Abe M, Ichimaru N, Harada T, et al. (2007) Crucial structural factors and mode of action of polyene amides as inhibitors for mitochondrial NADH-ubiquinone oxidoreductase (complex I). Biochemistry 46: 10365–10372.
- 50. Hatefi Y, Stiggall DL (1978) Preparation and properties of succinate: ubiquinone oxidoreductase (complex II). Methods Enzymol 53: 21–27.