Inhibition, Mitochondrial complex III

The mitochondrial complex III, also known as cytochrome bc1 complex, is located in the inner mitochondrial membrane. It is the third of the five complexes that form the mitochondrial respiratory chain. It contains three catalytic subunits, cytochrome b/b6, cytochrome c1 and the Rieske iron–sulfur protein an (2Fe-2S cluster). 

Complex III catalyzes electron transfer from ubiquinol (QH2) deriving from complex I, complex II and other mitochondrial systems, to cytochrome c. The complex has two functional binding sites for quinone, one is the oxidation site of quinone (QP or QO) on one side of the intermembrane space, close to heme b. The other is the quinone reduction site (QN or QI) on the matrix side, close to heme bH (Hunte et al, 2003). 

The electron transfer in this process mediates the translocation of protons from the mitochondrial matrix through the inner membrane to the intermembrane space. The created proton gradient will be used to catalyze the reaction in which ADP is converted into ATP. 

In functional mitochondria, Complex III can be found as free complex or in association with Complex I and Complex IV (CI-CIII2-CIV) in supramolecular structures called respirasomes (Letts & Sazanov, 2017). 

The initiation event is the reversible or irreversible interaction to any of the subunits in the mitochondrial complex III, leading to an perturbation of the electron flow and an absence of proton transport via this complex. 

In most cases, inhibition of Complex III occurs upon binding of inhibitors to one of the two quinone binding sites. Specific binding to the quinol oxidation site (QP or QO site), blocks the motion of the iron-sulfur protein subunit. Binding to this site confers to inhibitors selectivity between mammalian vs protozoan (Esser et al, 2023) or pests Complex III (Luo et al, 2022). 

Activity of complex III of the respiratory chain can be determined indirectly by the detection of oxygen consumption as a readout for the activity of oxidative phosphorylation in entire mitochondria. Direct enzymatic activity measurements include spectrophotometric assays. Detection of oxygen consumption is conducted either in permeabilized cells to allow unrestricted access of substrates and inhibitors to mitochondria or in isolated mitochondria. Direct enzymatic assays are performed in isolated mitochondria, or submitochondrial particles. 

Detection of complex III activity in isolated mitochondria or in submitochondrial particles by spectrophotometry. 

The decylubiquinol/cytochrome c reductase assay is the standard spectrophotometric method to measure the enzymatic activity of mitochondrial complex III (ubiquinol–cytochrome c reductase) (Krähenbühl et. al, 1994). The principle of the assay is based on the physiological role of complex III, which catalyzes the transfer of electrons from ubiquinol (QH₂) to cytochrome c in the respiratory chain. In the assay, the artificial substrate decylubiquinol (DB), a soluble analog of ubiquinol with a shortened aliphatic side chain, serves as the electron donor (Xia et al., 2013). Decylubiquinol is oxidized by complex III at the Qo site of cytochrome b, and the released electrons are transferred sequentially through the Rieske iron–sulfur protein and cytochrome c₁ subunit to the terminal electron acceptor, cytochrome c. The reduction of cytochrome c results in a characteristic increase in absorbance at approximately 550 nm, which can be continuously monitored using a spectrophotometer. The rate of cytochrome c reduction is directly proportional to the activity of complex III in the sample (Krähenbühl et. al, 1994). To ensure specificity, the assay is performed in the presence and absence of antimycin A, a potent inhibitor of the Qi site of complex III (Bujan et al., 2022). The difference between the total rate and the antimycin-insensitive rate defines the antimycin-sensitive activity, which corresponds to the true activity of complex III. This control is essential, as cytochrome c can also be reduced by other mitochondrial enzymes or non-specific redox reactions. 

Complex III activity detection is conducted according to the reaction: 

DB (reduced) + cytochrome c (oxidized) → DB (oxidized) + cytochrome c (reduced) (550 nm) 

The reaction is measured in a buffer consisting of: 50 mM potassium phosphate, pH 7.4, 1 mM n-dedecylmaltoside, 1 mM KCN, 2,5 µM rotenone, and 0.1 fatty acid free BSA (fraction V) . (Bujan et al., 2022; Krähenbühl et al., 1994). 

The sample is added to the reaction buffer, and allowed to equilibrate for a few minutes at 30 °C. Then, reduced decylbenzylquinone is added (100 µM), in parallel a blank sample without reduced DB is prepared. The reaction is initiated by the addition of cytochrome c (15 µM), absorbance is detected after a lag phase of ca. 20 sec. in 30 sec. intervals over a period of 3-5 min. Absorbance is measured at 550 nm. At the end of the recordings, an excess amount of ascorbic acid (50 mM) for full cytochrome c reduction is added, the resulting absorbance value is detected separately.  

The results are calculated as first-order rate constants as the reactions rapidly become non-linear. The final absorbance reading (ascorbic acid) are subtracted from each data point. The data are then plotted as a log plot against time, and the slope is the first order rate constant. (Bujan et al., 2022; Krähenbühl et al., 1994). 

Complex III activity detection in permeabilized cells or in isolated mitochondria based on the detection of oxygen consumption. 

The principle of oxygen detection by a Clark electrode is based on the electrochemical reduction of oxygen at a cathode, typically composed of platinum, which is maintained at a fixed potential relative to a reference electrode. The electrode system is immersed in an electrolyte solution and separated from the sample medium by an oxygen-permeable membrane, commonly made of Teflon or polyethylene. Oxygen from the sample diffuses through the membrane into the electrolyte, where it undergoes reduction at the cathode surface according to the reaction: 

O₂ + 4e⁻ + 4H⁺ → 2H₂O (in acidic medium) or 

O₂ + 2H₂O + 4e⁻ → 4OH⁻ (in neutral or alkaline medium). 

The rate of oxygen diffusion through the membrane, and thus the magnitude of the resulting cathodic current, is directly proportional to the partial pressure or concentration of dissolved oxygen in the sample (Li et al. 2012; Silva et al. 2012). This current is measured and converted into an electrical signal, which can be quantitatively related to oxygen concentration. To ensure accuracy and stability, the electrode is often maintained at a constant temperature, and the membrane thickness and electrolyte composition are carefully controlled. The Clark electrode, by providing real-time and continuous measurements, is widely used in biomedical, environmental, and industrial applications for monitoring oxygen levels. 

Optical oxygen sensors operate on the principle of dynamic quenching of luminescence by molecular oxygen. These sensors typically utilize oxygen-sensitive fluorophores or phosphorescent dyes immobilized within an oxygen-permeable matrix (Melnikov et al. 2022; Dmitriev et al. 2012). In the absence of oxygen, the fluorophores exhibit a characteristic luminescence—either fluorescence or phosphorescence—with defined intensity and lifetime upon excitation by a specific wavelength of light. When molecular oxygen is present, it acts as a dynamic quencher, reducing the luminescence intensity and/or shortening the emission lifetime through a collisional quenching mechanism governed by the Stern–Volmer relationship. The degree of quenching is quantitatively related to the local oxygen concentration, enabling precise and non-invasive measurements. Unlike electrochemical sensors, optical oxygen sensors do not consume oxygen during detection, making them particularly suitable for applications requiring high spatial and temporal resolution under physiologically relevant conditions. Extracellular flux analyzers such as the Seahorse system emerged as the frontline technique in high content oxygen consumption detection (Plitzko et al. 2018). Optical oxygen sensors are integrated into the bottom of specialized microplates used for real-time measurement of cellular respiration. Fluorescent or phosphorescent oxygen-sensitive dyes are embedded in solid-state sensor patches adhered to each well. During an assay, a transient microchamber is formed above the adherent cells by lowering a sensor cartridge, temporarily isolating a small volume of medium. The consumption of oxygen by the cells within this microenvironment results in a measurable decrease in oxygen concentration, which is detected by changes in the fluorescence signal of the sensor. 

This real-time, label-free detection enables the calculation of the oxygen consumption rate (OCR), a key parameter reflecting mitochondrial respiratory activity. When coupled with simultaneous measurement of extracellular acidification rate (ECAR), these data provide comprehensive insights into cellular bioenergetics, metabolic reprogramming, and mitochondrial function in live cells under various physiological and pharmacological conditions (Yoo et al. 2024; Zhang et al. 2019). 

Due to its widespread use in current research and its applicability on a high-content scale, the following descriptions will consistently refer to the use of the fluorophore-based method. 

Cell permeabilization 

Controlled permeabilization of cells allows access to mitochondria in their natural environment without the time-consuming isolation procedures that affect mitochondrial function. The principle of cell permeabilization by agents such as digitonin and saponin is based on their ability to selectively disrupt the plasma membrane while preserving the integrity and functionality of intracellular organelles, particularly mitochondria. This selective permeabilization is essential for enabling the controlled introduction of exogenous substrates, ADP, and specific inhibitors directly to mitochondria within their native cellular context, thereby allowing accurate measurement of mitochondrial respiration (Salabei et al. 2014). Digitonin and saponin are amphipathic glycosides that interact preferentially with cholesterol-rich domains of biological membranes. The plasma membrane, which contains a relatively high cholesterol content, is particularly susceptible to disruption by these agents. In contrast, the mitochondrial inner membrane contains little cholesterol and is largely resistant to their action at appropriately optimized concentrations. When cells are treated with low concentrations of digitonin or saponin, the plasma membrane becomes permeable, allowing small molecules (e.g., metabolic substrates, ADP, respiratory inhibitors) to diffuse into the cytoplasm (Divakaruni et al. 2014; Niklas et al. 2011; Lei et al. 2021). This creates a semi-intact cell model in which mitochondrial membranes remain functionally intact, and mitochondrial respiratory activity can be assessed in a highly controlled, substrate-specific manner. Importantly, the preservation of mitochondrial integrity ensures that electron transport, membrane potential, and ATP synthesis can be reliably studied. Selective permeabilization using digitonin or saponin provides an experimental platform, bridging the gap between isolated mitochondria and intact cells, and is particularly compatible with high-resolution platforms like the Seahorse XF Analyzer. 

Complex III activity in permeabilized cells 

In the 96 well format, a cell density in the range of 30.000-80.000 cells per well (depending on the cell type) is added in a volume of 80 µl and centrifuged (200 g for 1 min) to ensure attachment on the bottom of the plate. Depending on the cell type, the cells are then allowed to attach for a certain time period (e.g. 24 h) to ensure proper attachment. If cells in suspension are employed, the measurements can be initiated immediately. Following treatment of the cells, permeabilization is initiated according to the protocols described in Salabei (2014) or Delp (2019). In brief, permeabilization is initiated by MAS buffer (220 mM mannitol, 1 mM ADP, 70 mM sucrose, 10 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, 1 mM EGTA, 4 mg/ml fatty acid free BSA, and a permeabilizing agent such as digitonin at a concentration of 20 – 200 µg/ml, pH 7.2. The optimal concentration of the permeabilizing agent is highly dependent on the cell type and cell density and needs to be empirically determined for each individual setup. Higher concentrations ensure effective permeabilization, however at the same time harm the integrity and function of mitochondria. At a digitonin concentration of 100 µg/ml, measurement of oxygen consumption rate (OCR) can be initiated after ca. 5 min. Due to breakdown of cytosolic integrity, basal mitochondrial respiration proceeds on a relatively low level under these conditions (Djafarzadeh & Jakob, 2017; Makrecka-Kuka et al., 2015).  

Fig. 1 Mitochondrial respiratory chain, detection of complex III activity. In permeabilized cells, respectively in isolated mitochondria, the overview illustrates the specific input of electrons into the respiratory chain by selected substrates. For the determination of complex III activity, inhibitors of complex I and complex II are added first to prevent electron influx via these complexes originating from residual NADH or FADH2 formation in the cells. Then, the complex III selective electron donor duroquinol is added. The observed rise in OCR directly reflects complex III activity as all other electron input sites are blocked under these conditions. (Toki et al. 2022).

Complex III activity determination requires the addition of complex I and complex II inhibitors. This step ensures that no electron influx, originating from residual NADH or FADH2 from the cell, contributes to oxygen consumption. Then, the complex III substrate duroquinol is added and results in an increase in OCR. If a test compound that inhibits complex III is present, the addition of duroquinol results in an impaired rise in the OCR. If test compounds show an inhibitory effect on OCR following duroquinol addition, it is essential to include testing of complex IV activity. If complex IV is not affected, the conclusion of complex III inhibition can be made (Divakaruni et al., 2022).  

Fig. 2 Exemplary result of complex III detection by an extracellular flux analyzer. Oxygen consumption rate (OCR) detection was started 5 min after addition of permeabilizer (t =0). As test compounds, a complex III inhibitor (white) as well as a compound that does not interfere with complex III (black) were added. Then, complex III substrate duroquinol is added, leading to an increase in OCR. To exclude a contribution of complex IV to the observed inhibition, the selective determination of complex IV activity in a separate experiment is mandatory (Illustrative example provided by Schildknecht S). 

Taxonomic: Some sections of taxonomic description were derived from the ENV/JM/MONO(2020)23 Series on testing and assessment no. 327 (Bennekou et al. 2020) 

Complex III is a component of the aerobic respiratory chain found in both prokaryotic and eukaryotic organisms. 

Complex III is highly conserved among mammals and less conserved vs protozoan, fungi and bacteria (Table 1), especially regarding the QP or QO site (Xia et al. 2013; Trumpower et al. 1990; Dibrova et al. 2013; Fisher et al. 2020; Vincent et al. 2016; Esser et al. 2024). Although the catalytic core of cIII (cytochrome b, cytochrome c₁, and the iron-sulfur protein) is structurally and functionally conserved across species, its overall composition and regulatory mechanisms vary among taxa. In bacteria, cIII contains only the core subunits and structural insertions that aid in stability and lipid interaction (Xia et al. 2013; Trumpower et al. 1990). Yeast and mammals possess additional supernumerary subunits that enhance structural integrity and facilitate complex assembly (Xia et al. 2013). Plants uniquely integrate mitochondrial processing peptidase activity into their cIII, combining electron transport with protein processing (Xia et al. 2013; Maldonado et al. 2021; Xia et al. 2013). 

Functional adaptations include species-specific insertions in cytochrome b and c₁, variations in ISP mobility, and differences in lipid and metal ion interactions (Xia et al. 2013). These modifications contribute to enhanced stability, regulatory control, and integration into respiratory supercomplexes. Numerous structural and functional studies have been performed on Complex III isolated from different animal species, e.g. H. sapiens, M. musculus, R. norvegicus, D. rerio (Guo et al. 2017; Xia et al. 2025; Huang et al. 2005; Xia et al. 2013; Garcia-Poyatos et al. 2020; Buck et al. 2014).  Subtle differences among mammalian species could be ascribed to the predominant quinone species, characterized by differences in the isoprenoid chain length (Lass et al. 1997; Olgun et al. 2003; Pallotti et al. 2021).  

Table 1 reports the percentage of identity of inhibitor-binding subunits obtained by amino acid comparison.  

Table 1. Percentage of identity of mitochondrial cIII inhibitor-binding subunit (cytochrome b) 

Protein BLAST analysis of S. cerevisiae has shown high matches for residues that compose the catalytic core of complex III at the binding site for strobin inhibitors on Qo, and on the respective haeme bH binding site, as well as on Qi and the respective haeme bL binding site (Bennekou et al 2020). 

The differences in cytochrome b-Qo (which binds myxothiazol and atovaquone) and cytochrome b-Qi (which binds antimycin) are functionally relevant, since antimycin displays an LC50 value that is over 1000 times lower towards zebrafish and mammals than towards parasites (Pinho et al. 2013). Atovaquone is much more toxic to P. falciparum than to zebrafish and mammals (Pinho et al. 2013). 

Comparison of the available crystal structures for CIII of yeast and vertebrates did not show features that would interfere with the coupling process of proton pumping and electron transfer of the Q-cycle mechanism (Berry, De Bari, & Huang, 2013). 

Life stage: Given the essentiality of complex III in mitochondrial respiration, mitochondrial function and cell homeostasis, it is inferred that its inhibition is detrimental at all life stages.  

Sex: Sex differences in mitochondrial respiratory complex activity are evident across various tissues and life stages in mammals. Data for an explicit sex-dependent influence of complex III however are limited. A meta-analysis of human studies on mitochondrial biology – including complex III activity – indicates lack of binary sex differences in mitochondrial biology (Junker et al, 2022). 

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