Coenzyme Q (Q, ubiquinone or 2,3-dimethoxy-5-methyl-6-polyprenyl-1,4-benzoquinone) is a ubiquitous fat-soluble vitamin-like compound distributed in all cell membrane systems, including the plasma membrane and all intracellular membranes, primarily the inner mitochondrial membrane [4, 5, 6]. Q consists of a redox active benzoquinone ring with two methoxy groups, one methyl group, and an isoprenoid side chain with a species-specific length (6 to 10 units) (Fig. 2). For example, ten isoprenoid units (Q10) have been described as the most commonly found Q in humans and nine units (Q9) in rodents (Q6) [7]. Some species have more than one Q isoform, e.g., rodent tissues contain different proportions of Q9 and Q10. In cells, Q is present in three different redox states, i.e., fully oxidized (ubiquinone, Q), intermediate semioxidized (semiubiquinone radical, QH${}^{\mathrm{•}}$), and fully reduced (ubiquinol, QH${}_2$) (Fig. 2) [8, 9, 10]. Thus, Q has the ability to accept and transfer one or two electrons, which is crucial for its role as a one-electron carrier in the mitochondrial electron transport chain because of the iron-sulfur (FeS) clusters, which can only accept one electron at a time [11], and for its role as a powerful antioxidant scavenging free radicals [12, 13].

Fig. 2.

Q redox states. Oxidized form of Q (ubiquinone) can be reduced to ubiquinol (QH${}_2$) by two one-electron reactions through semiubiquinone (QH${}^{\mathrm{•}}$) intermediate, or by one reaction of two electrons. Superoxide (O${}_2$${}^{\mathrm{•}\mathrm{¯}}$) and hydrogen peroxide (H${}_2$O${}_2$) can be produced by the leakage of electrons from the redox group of QH${}^{\mathrm{•}}$ with unpaired electron.

Unlike other lipophilic antioxidants such as vitamin A, carotenoids, and vitamin E, which are usually obtained from dietary sources, Q can also be produced endogenously [14]. The synthesis of Q takes place in two main steps, one of which provides the quinone ring, while the other produces the isoprenoid tail (for reviews see [15, 16, 17, 18, 19, 20]). The 4-benzoquinone ring is synthesized by converting tyrosine (or phenylalanine) to 4-hydroxybenzoate, while the polyprenyl tail is provided via the mevalonate pathway, which also synthesizes cholesterol and dolichols, i.e., large isoprenoid chains comprised of multiple isoprene units. The synthesis of Q precursors (4-hydroxybenzoate and polyprenylpyro-phosphate) takes place in the cytosol, and the last stages of Q biosynthesis take place on the matrix side of the inner mitochondrial membrane [21, 22]. The quinone head is attached to a polyprenylated tail by a prenyl transferase (COQ2) which, together with other enzymes, forms a multiprotein Q biosynthetic complex on the matrix side of the inner mitochondrial membrane [7, 21]. After multiple modifications of the aromatic ring, including methylation, decarboxylation, hydroxylation and deamination (catalyzed by COQ3, COQ5, COQ6, and COQ7, respectively), the final Q molecule is formed. Defects in Q synthesis, including the highly regulated mitochondrial biosynthetic complex, can lead to a reduction in mtQ levels and thus to disturbance of mtQ redox homeostasis and oxidative stress [23].

Despite the vast number of publications on both cellular and mitochondrial Q, its antioxidant and bioenergetic functions, role in health and disease, the literature is surprisingly limited regarding the relationship between Q redox state and ROS production in mitochondria under physiological and pathological conditions. In addition, the role of the mtQ redox state (mQH${}_2$/mtQ ratio) is poorly understood because determining its value is technically difficult. In this review, we focus on factors affecting mtQ redox homeostasis and its relationship to mtROS production. We first describe the mtQ-reducing and mtQ-oxidizing pathways of the mitochondrial electron transport chain and the factors influencing the redox state or reduction level of the mtQ pool. We then discuss the relationship between mtQ and mtROS formation, with an emphasis on the mtQ-biding sites of mtROS production, the dependence of mtROS formation on mtQ reduction level, and the relationship between mtQ content and mtROS production. Finally, we provided an introduction to the role of reduced mtQ as an antioxidant. In conclusions, we describe the technical progress in determining the redox state of Q in cells, tissues and isolated mitochondria.

mtQ is reduced to mtQH${}_2$ by several dehydrogenases that oxidize reducing equivalents derived from various food fuels, i.e., glucose, fats and amino acids [29]. Thus, mtQ is a common reservoir of electrons that contribute to oxidative oxidative phosphorylation, regardless of source.

Complex I and complex II of the mitochondrial respiratory chain reduce mtQ pool with electrons from the reduced nucleotides (NADH and FADH${}_2$, respectively) derived mainly from the TCA cycle and thus from the oxidation of major food molecules. In the rotenone-sensitive complex I, two electrons are transferred from NADH to the flavin mononucleotide (FMN) and then, through the iron-sulfur centers to mtQ, which is reduced to mtQH${}_2$ [30, 31, 32]. The malonate-sensitive complex II is an enzyme involved in the TCA cycle and oxidizes succinate to fumarate. Complex II transfers two electrons from FADH${}_2$ through the iron-sulfur centers to mtQ, thereby also introducing two electrons into the mitochondrial respiratory chain [33, 34, 35].

In addition to the basic dehydrogenase complexes of the respiratory chain, i.e., complex I and complex II, mtQ receives electrons from a number of electron transporting flavoproteins that supply electrons to the mtQ pool [36, 37, 38]. mtQ is reduced by electron transfer flavoprotein (ETF):mtQ oxidoreductase (ETF:mtQ oxidoreductase) that oxidizes FADH${}_2$ derived from $\beta$-oxidation of fatty acids in the mitochondrial matrix [39, 40]. Electrons are also delivered to the mtQ pool from the oxidation of dihydroorotate to orotate by dihydroorotate dehydrogenase, a key mitochondrial enzyme in pyrimidine nucleotide biosynthesis [41, 42]. Another mtQ-reducing dehydrogenase located on the outer surface of the inner mitochondrial membrane is FAD-linked glycerol-3-phosphate dehydrogenase, which catalyzes the conversion of glycerol-3-phosphate to dihydroxyacetone [43, 44]. The oxidation of glycerol-3-phosphate by mitochondrial glycerol 3-phosphate dehydrogenase is a major pathway for the transfer of cytosolic reducing equivalents from lipid and carbohydrate metabolism to the mitochondrial electron transport chain. In addition, mtQ accepts electrons from FAD-linked proline dehydrogenase, which is involved in the metabolism of amino acids [45, 46]. In the branched mitochondrial respiratory chain of plants, fungi, and some protists, mtQ receives electrons from alternative rotenone-resistant NAD(P)H dehydrogenases located on the outer and inner surfaces of the inner mitochondrial membrane [47, 48]. Among the mtQ-reducing dehydrogenases, only complex I of mitochondrial respiratory chain pumps protons and therefore participates in the generation of the proton electrochemical gradient.

mtQ metabolism depends on mtQH${}_2$ reoxidation by the mitochondrial mtQH${}_2$-oxidizing pathway(s). In mammalian mitochondria, the cytochrome pathway (complex III, cytochrome c, and finally complex IV) is the only mtQH${}_2$-oxidizing pathway in the respiratory chain [2, 27]. The antimycin A- and myxothiazol-sensitive complex III and the cyanide-sensitive complex IV pump protons through the inner mitochondrial membrane to the intermembrane space and therefore participates in the generation of the protonmotive force. In the branched mitochondrial respiratory chain of plants, fungi, and some protists, there is an additional antimycin A- and cyanide-resistant alternative quinol oxidase (AOX) that oxidizes mtQ, bypassing the cytochrome pathway [49, 50]. Transfer of electrons in AOX is not accompanied by proton pumping and thus mitochondrial ATP production.

Complex III (mtQH${}_2$:cytochrome c oxidoreductase, cytochrome bc${}_{\mathrm{1}}$ complex) of the cytochrome pathway oxidizes the mtQH${}_2$ pool directly, catalyzing a series of electron transfer reactions from mtQH${}_2$ to cytochrome c, called the mtQ cycle (Fig. 1, marked box) [51, 52, 53]. Thus, complex III connects two mobile electron carriers of the respiratory chain, mtQ and cytochrome c. The reducing force of the mtQH${}_2$ pool and the oxidizing force of the cytochrome c pool influence the redox state of the four metal redox centers in complex III: hemes b${}_{\mathrm{\text{H}}}$ and b${}_{\mathrm{\text{L}}}$ in cytochrome b, and heme c${}_{\mathrm{1}}$ and the Rieske FeS center in cytochrome c${}_{\mathrm{1}}$. mtQ reduction and mtQH${}_2$ oxidation reactions take place at two separate catalytic centers. The myxothiazol-sensitive Q${}_{\text{o}}$ site is the site of binding and oxidation of QH${}_2$ located toward the intermembrane space, while the antimycin A-sensitive Q${}_{\text{i}}$ site is the site of binding and reduction of mtQ located on the matrix side.

The mtQ cycle of complex III involves all three forms of Q (fully oxidized, semioxidized, and fully reduced) (Fig. 3) and participates in the generation of the protonmotive force as during mtQH${}_2$ oxidation protons are released into intermembrane space [51, 52, 53, 54]. The mtQ cycle involves a series of one-electron redox reactions as a result of bifurcation of electrons at the Q${}_{\text{o}}$ site (Fig. 1, marked box). At this site, one electron from mtQH${}_2$ is transferred to the Rieske protein, then to cytochrome c${}_{\mathrm{1}}$, and finally to cytochrome c; the second electron is translocated through heme b${}_{\mathrm{\text{H}}}$ and heme b${}_{\mathrm{\text{L}}}$ to the catalytic site Q${}_{\text{i}}$, where mtQ is reduced to a semiubiquinone radical (QH${}^{\mathrm{•}}$) [51, 52, 53, 54, 55]. The oxidation of two mtQH${}_2$ molecules involves the reduction of two cytochrome c molecules and completes the reduction of semiubiquinone to mtQH${}_2$ at the Q${}_{\text{i}}$ site. Thus, a complete enzymatic cycle of complex III gives the oxidation of one mtQH${}_2$ with the reduction of two molecules of cytochrome c and the return of half of the electrons within the mtQ pool.

Fig. 3.

Factors influencing the mtQ reduction level. mtQ, oxidized mitochondrial coenzyme Q; mtQH${}_2$, reduced mtQ, quinol; mtQ${}_{\text{tot}}$, total mtQ (mtQ + mtQH${}_2$) in the inner mitochondrial membrane.

Although one of the main functions of the mtQ-reducing and mtQH${}_2$-oxidizing pathways is to maintain the mtQ redox homeostasis, there are few studies describing a direct relationship between their activity and the level of mtQ reduction (or mtQ redox state) and mtROS production under physiological and pathological conditions.

In the inner mitochondrial membrane, mtQ is a molecule directly involved in the production of mtROS due to the formation of O${}_2$${}^{\mathrm{•}\mathrm{¯}}$/H${}_2$O${}_2$ by the leakage of electrons from the semiubiquinone radical. All four mtQ-binding sites (I${}_{\text{Q}}$, III${}_{\text{Qo}}$, G${}_{\text{Q}}$, D${}_{\text{Q}}$) produce O${}_2$${}^{\mathrm{•}\mathrm{¯}}$ and only one of them (D${}_{\text{Q}}$) is likely to produce H${}_2$O${}_2$ as well [80, 85].

At I${}_{\text{Q}}$ of complex I, O${}_2$${}^{\mathrm{•}\mathrm{¯}}$ is mainly generated during reverse electron transport (RET), i.e., a transfer of electrons against the redox potential gradient supported by a high proton electrochemical gradient (a high protonmotive force) across the inner mitochondrial membrane and a high mtQ redox state (highly reduced mtQ pool) [86]. Under these conditions, mtQH${}_2$ is oxidized to mtQ at the mtQ-binding site (I${}_{\text{Q}}$) to drive the reduction of NAD${}^{+}$ to NADH at the FMN site of complex I (I${}_{\text{F}}$). Inhibition of mtQ-binding with rotenone inhibits RET and lowers mtROS production.

At the outer mtQ-binding site of complex III (III${}_{\text{Qo}}$), O${}_2$${}^{\mathrm{•}\mathrm{¯}}$ production is low in the absence of Q${}_{\text{i}}$ site inhibitors [94]. In the presence of antimycin A, which binds to the Q${}_{\text{i}}$ site and blocks the oxidation of cytochrome b${}_{\mathrm{\text{L}}}$ heme, the mtQ cycle is disrupted. The resulting accumulation of reduced cytochrome b${}_{\mathrm{\text{L}}}$ heme limits semiubiquinone oxidation at site Q${}_{\text{o}}$. Semiubiquinone interacts with oxygen to form O${}_2$${}^{\mathrm{•}\mathrm{¯}}$ on both sides of the inner mitochondrial membrane. In the absence of antimycin A, a high protonmotive force and highly reduced mtQ pool decreases the electron transfer from the cytochrome b${}_{\mathrm{\text{L}}}$ heme to the cytochrome b${}_{\mathrm{\text{H}}}$ heme, leading to more reduced b${}_{\mathrm{\text{L}}}$ heme and thus increased O${}_2$${}^{\mathrm{•}\mathrm{¯}}$ production [95].

Site G${}_{\text{Q}}$ of mitochondrial glycerol-3-phosphate dehydrogenase produces O${}_2$${}^{\mathrm{•}\mathrm{¯}}$ in approximately equal amounts on each side of the mitochondrial inner membrane, suggesting that the mtQ-binding site of the enzyme is the major site of O${}_2$${}^{\mathrm{•}\mathrm{¯}}$ formation during glycerol-3-phosphate oxidation [87]. Dihydroorotate dehydrogenase can generate O${}_2$${}^{\mathrm{•}\mathrm{¯}}$ and/or H${}_2$O${}_2$ directly at its mtQ-binding site (D${}_{\text{Q}}$) at low rates [88]. The enzyme is also capable of indirect production at higher rates from other sites through its ability to reduce the mtQ pool.

In a cell, the net O${}_2$${}^{\mathrm{•}\mathrm{¯}}$/H${}_2$O${}_2$formation rate at mitochondrial mtQ-binding sites depends on the substrates being oxidized and the antioxidant systems acting on both sides of the inner mitochondrial membrane. Among the mtQ-binding sites, III${}_{\text{Qo}}$ and I${}_{\text{Q}}$, which is mainly active during RET, dominate in O${}_2$${}^{\mathrm{•}\mathrm{¯}}$ production and play an important role in redox signaling and the induction of oxidative damage [80].

As a mobile component of the mitochondrial electron transport chain, mtQ acts as a pro-oxidant in the semiubiquinone state. It is generally accepted that the production of mtROS depends on the level of reduction of the mtQ pool, which determines the formation of O${}_2$${}^{\mathrm{•}\mathrm{¯}}$ from semiubiquinone. However, there are only a few data relating the mtQ redox state, i.e., mtQH${}_2$/Q ratio [96] and mtQH${}_2$/mtQ${}_{\text{total}}$ ratio [59, 66, 68, 95], with mtROS generation. The formation of mtROS related to the activity of the respiratory chain, and thus the mtQ redox state, depends on the activity of all mtQ-reducing dehydrogenases and mtQH${}_2$-oxidazing pathway(s), and on the activity of the oxidative phosphorylation process in which ATP is formed [59]. We have shown that under ADP phosphorylating conditions (state 3), a low mtQ reduction level and a low m$\mathrm{\Delta }$$\mathrm{\Psi }$ are accompanied by decreased mtROS production [59]. In a nonphosphorylating state (state 4), a highly reduced mtQ pool (a high mtQ reduction level) and a high m$\mathrm{\Delta }$$\mathrm{\Psi }$ are accompanied by increased mtROS production. Thus, the mtQH${}_2$/mtQ ratio is sensitive to changes in electron supply from reducing equivalents and to ATP demand. The activity of uncoupling proteins (UCPs), which mediate proton leakage, can lower m$\mathrm{\Delta }$$\mathrm{\Psi }$ and the mtQ reduction level, leading to a decrease in mtROS production [76, 84].

The relationship between mtROS production and mt$\mathrm{\Delta }$$\mathrm{\Psi }$ was investigated more often than the interplay between mtROS formation and the mtQ reduction level. For the first time, the dependence of mtROS formation on mt$\mathrm{\Delta }$$\mathrm{\Psi }$ was described in rat heart mitochondria [97]. Namely, a very steep dependence of H${}_2$O${}_2$ formation on mt$\mathrm{\Delta }$$\mathrm{\Psi }$ was observed in nonphosphorylating rat heart mitochondria after exceeding a threshold value near mt$\mathrm{\Delta }$$\mathrm{\Psi }$ of ADP phosphorylating mitochondria. However, not under all conditions lowering mt$\mathrm{\Delta }$$\mathrm{\Psi }$ leads to a decrease in mtROS production (e.g., in the presence of inhibitors of complexes III and IV), indicating that mtROS is not a direct function of the mt$\mathrm{\Delta }$$\mathrm{\Psi }$ level (Fig. 5A) [59]. For example, we have shown that in the mitochondria of the amoeba Acanthamoeba castellanii, with inhibition of the Q${}_{\text{i}}$ site of complex III by antimycin A or complex IV by cyanide, m$\mathrm{\Delta }$$\mathrm{\Psi }$ decreases while the mtQ reduction and H${}_2$O${}_2$ production increase [59]. Marphy’s group has shown that the formation of O${}_2$${}^{\mathrm{•}\mathrm{¯}}$ at the Q${}_{\text{o}}$ site is associated with the accumulation of reduced cytochrome b${}_{\mathrm{\text{L}}}$ when the Q${}_{\text{i}}$ site is blocked by antimycin A, and this phenomenon is not dependent on mt$\mathrm{\Delta }$$\mathrm{\Psi }$ [95].

Fig. 5.

The relationship between mtROS formation versus mt$\mathrm{\Delta }$$\mathrm{\Psi }$ and the mtQ reduction level. The relationship between H${}_2$O${}_2$ formation versus mt$\mathrm{\Delta }$$\mathrm{\Psi }$ (A) for the cytochrome pathway (CII + CIII + CIV) fueled by complex II (CII) and the relationship between H${}_2$O${}_2$ formation versus the mtQ reduction level (mtQH${}_2$/mtQ${}_{\text{tot}}$) (B) for the CII + CIII + CIV pathway (blue curves and lines) and the CII-fueled alternative oxidase (AOX) (CII + AOX) (purple line). Based on results described in A. castellanii mitochondria [59]. The mt$\mathrm{\Delta }$$\mathrm{\Psi }$ level (A) and mtQ reduction level (B) of the CII + CIII + CIV pathway was varied with the ATP synthesis and CII inhibitors, with the uncoupler (blue solid curves), and with the inhibitor of CIII (antimycin A, AA) (blue dashed lines) when AOX inhibitor was present. Measurements were carried out under nonphosphorylating (state 4) and phosphorylating (state 3) conditions. (B) For the CII + AOX pathway, the mtQ reduction level was titrated with an activator of AOX (GMP) and an inhibitor of CII (malonate) in the presence of AA to block the cytochrome pathway.

We have kinetically described a direct dependence of mtROS production on the mtQ reduction level in A. castellanii mitochondria [59]. For both mtQH${}_2$-oxidizing pathways (the cytochrome and AOX pathways), during the oxidation of succinate fueling the respiratory chain at the level of complex II, a higher level of mtQ reduction leads to greater mtROS formation (Fig. 5B) [59]. This relationship is also observed for the cytochrome pathway in the presence of antimycin A, which by inhibiting the Q${}_{\text{i}}$ site lowers m$\mathrm{\Delta }$$\mathrm{\Psi }$ and increases mtQ reduction level. In the case of the cytochrome pathway, H${}_2$O${}_2$ generation depends nonlinearly on the reduction level of mtQ pool. At levels of mtQ reduction higher than that of the phosphorylating state (above 35%) there is a steep relationship between H${}_2$O${}_2$ generation and the mtQ reduction level. In contrast, AOX is active only when the mtQ reduction level is high (above 40%) and the dependence between H${}_2$O${}_2$ generation and mtQ reduction level is approximately linear (Fig. 5B). We have proposed that the mtQ pool reduction level (the endogenous redox state of mtQ) may be a useful endogenous marker that allows for the assessment of total mitochondrial mtROS production [59].

High levels of mtROS are generated in response to stress via RET through complex I [61, 98]. RET occurs when the mtQ pool it is strongly reduced by electrons from the respiratory complex II (succinate dehydrogenase) [95, 98]. In various cell lines, it has been observed how the respiratory chain is optimized for better oxidation of various reducing respiratory fuels by the redox state of mtQ (mtQH${}_2$/Q ratio) acting as a metabolic detector and mtROS formed by complex I (via RET) acting as an executor [96]. In the rat heart mitochondria, the formation of mtROS by RET at complex I has been shown to be dependent on the mtQ reduction level and mt$\mathrm{\Delta }$$\mathrm{\Psi }$ that indicates the sensitivity of O${}_2$${}^{\mathrm{•}\mathrm{¯}}$ formation by the mtQ-binding site of complex I to these two metabolic variables [95].

It seems that the production of mtROS does not directly depend on the amount of mtQ but rather on the reduction level of the mtQ pool (mtQH${}_2$/mtQ${}_{\text{total}}$ ratio) as a function of the activity of mtQ-reducing pathways (mtQ-reducing dehydrogenases) and the mtQH${}_2$-oxidizing cytochrome pathway(s). In the heart mitochondria of MCLK1 (CoQ7) mutant mice, which contain only ~10% of normal Q content, lower rates of mtROS production from all known mtROS production sites were detected at respiration levels similar to those in control mitochondria [99]. One possible explanation for how low mtQ levels reduce mtROS formation is that low mtQ induces the formation of supercomplexes that have been proposed to increase electron transport efficiency from one redox component to another, thus minimizing electron leakage and mtROS production [100, 101]. In cultured fibroblasts from Q-deficient patients, increased mtROS production was observed during intermediate Q deficiency (50–70%) possibly as a result of increased semiubiquinone generation from increased redox cycling of the restricted mtQ pool, while a more severe loss of Q ($>$85%) was not accompanied by significant mtROS production [102]. Interestingly, we have shown that in lung mitochondria of endurance-trained rats, reduced mtQ content (mtQ9 by ~16% and mtQ10 by ~42%) and decreased activity of complex I as well as increased activity of cytochrome pathway (complex III plus complex IV) may account for the observed diminished mtQ reduction level during succinate and/or malate oxidation, resulting in a general decrease of mtROS formation by mitochondria [66]. Moreover, endurance training downregulates complex I in supercomplexes and upregulates complex III in the CIII${}_2$ + CIV supercomplex in the inner membrane of lung mitochondria. In addition, comparison of the effects of endurance training on mtQ amount and mtROS generation in rat tissues with high energy demand, i.e., in the brain, liver and heart, indicates that endurance training may induce various mitochondrial and tissue responses related to mtQ acting as an electron carrier in the respiratory chain and as an antioxidant [67]. Changes in the formation of mtROS observed in the mitochondria of individual rat tissues may result from changes in the size of the pool of oxidized mtQ, which acts as an electron carrier, as well as the amount and activity of individual complexes of the oxidative phosphorylation system and its molecular organization.

A decrease in the mtQ pool size and mtQ reduction level may results from drug treatment. We have shown that in cultured endothelial cells, chronic exposure to atorvastatin, an inhibitor of HMG-CoA reductase led to a significant decrease in mtQ10 content (~23%) [68]. The activity of complex III and its amount in a supercomplex with complex IV was also diminished. The statin-induced changes at the respiratory chain level of the endothelial mitochondria led to a decrease in succinate and/or malate oxidation accompanied by a decreased mt$\mathrm{\Delta }$$\mathrm{\Psi }$, as well as to an increase in the mtQ10 reduction level and consequently increased mtROS production.

Thus, mtQ redox homeostasis is a key factor in modulating mtROS production. As mentioned above, depending on the activity of mtQ-reducing pathways and mtQH${}_2$-oxidizing pathways, a decreased amount of mtQ can lead to either an increased reduction level of mtQ (a higher mtQH${}_2$/mtQ${}_{\text{total}}$ ratio) and thus to an increased production of mtROS or to a decreased reduction level of mtQ (a lower mtQH${}_2$/mtQ${}_{\text{total}}$ ratio) and hence decreased production of mtROS. Superphysiological mtQ levels could possibly have the same effect by altering the level of mtQ reduction. Thus, changing the size of the mtQ pool and its redox state is an important physiological adaptation. The metabolic response to the altered mtQ pool appears to depend on whether it is more important to maintain an efficient oxidative phosphorylation process or whether it is more important to defend against oxidative stress.

The level of Q, including mtQ, varies with the type of membrane, tissue and organism [4, 103]. It is highest in organs with a high metabolic rate and hence a high energy requirement, such as the heart, muscles, and liver. However, despite high metabolic activity, total Q levels and the QH${}_2$/Q ratio in the human brain are very low compared to other tissues [4]. The mtQ pool is in excess compared to other components of the respiratory chain [100]. However, with age and in many disease states, including primary and secondary Q deficiencies, associated with increased production and action of mtROS, the level of cellular and thus mitochondrial Q decreases [23, 60, 71, 72, 104]. mtQ deficiency may impair oxidative phosphorylation and therefore respiratory chain activity and ATP synthesis [105, 106, 107, 108], and may induce oxidative stress (mtROS overproduction) leading to oxidative modification of macromolecules, and ultimately to apoptosis. However, the question of how Q deficiency due to disease or aging affect the redox state of mtQ has not yet been thoroughly investigated.

Increasing mtQ levels must also result in a change in mtQ redox homeostasis and mtROS production. For example, we have observed an increase in mtQ9 content (by ~20%) in skeletal muscle mitochondria after eight-week endurance training [54]. In these mitochondria, H${}_2$O${}_2$ production and mtQ reduction level were elevated under nonphosphorylating conditions, and decreased under phosphorylating conditions, while the efficiency of oxidative phosphorylation increased with unchanged components of the respiratory chain. Recently, it has been shown that elevated levels of mtQ caused by Q10 delivery (via by BPM31510) lead to an increase in ROS generation and consequently, an imbalance in cellular redox homeostasis that activates mtROS-mediated cell death pathways in pancreatic cancer cells [109]. Similarly, cell death activation due to increased mtQ pool was observed in vitro in pancreatic cancer cells, and both human patient-derived organoids and tumor xenografts. Interestingly, in other studies, chemotherapy induced increased production of ROS and increased levels of Q10 (especially its reduced form Q10H${}_2$) in all cancer cell lines tested [110]. But again, we do not know how mtQ redox state changes under these conditions and what role it plays in mtROS production.

We have proposed that the antioxidant effect may also be exerted by the reduced mtQH${}_2$ pool in the inner mitochondrial membrane, which is not oxidized by the respiratory chain [74]. Interestingly, in mitochondria isolated from different rat tissues with different energy requirements and different mitochondrial oxidative activity heart, brain, liver and lungs, the size of the mtQH${}_2$ pool that was not oxidized by the respiratory chain (measured in the absence of respiratory substrates) was associated with the mtROS production capacity during the oxidation of respiratory substrates [74]. Thus, the size of the reduced nonoxidizable pools of mtQ (nonoxidizable mtQH${}_2$ pool) may reflect the need for mtQ as a mitochondrial antioxidant. Therefore, it is possible that both the mtQH${}_2$ pool, which participates in the mtQ cycle of the electron transport chain, and the mtQH${}_2$ pool, which is not oxidized by the chain, contribute to the overall antioxidative activity of mitochondria.