Redox cycling of a chemical by mitochondria

Redox cycling is a process of alternate reduction and reoxidation steps. Redox cyclers are compounds capable of undergoing repeated cycles of reduction and oxidation, driving the formation of reactive oxygen species (ROS) such as superoxide (O₂•⁻). (Drechsel et al. 2009; Hassan et al. 1984; Bus et al. 1974) This process begins when a redox cycler accepts a single electron from a biological reductant—often catalyzed by flavoprotein oxidoreductases like NADPH-cytochrome P450 reductase, nitric oxide synthase, or mitochondrial enzymes such as NADH-cytochrome b5 reductase and components of the respiratory chain, particularly complex I (Shimada et al. 1998; Thor et al. 1982). The electron transfer to the redox cycler forms a transient radical intermediate. This radical is highly reactive and quickly donates its extra electron to molecular oxygen, forming superoxide and regenerating the parent compound. Because the parent redox cycler is restored, the cycle can repeat, continuously producing ROS as long as both reductant and oxygen are available (Fig. 1). 

Fig. 1. Schematic representation of the mechanism of chemicals redox cycling. (Adapted by permission from Macmilllan Publishers Ltd, Cohen and Doherty, 1987, copyright (1987)) 

The efficiency and direction of redox cycling depend on the redox potential of the involved species. Redox cyclers typically have highly negative reduction potentials, enabling them to accept electrons from specific cellular reductants and to rapidly reduce oxygen (standard redox potential for O₂/O₂•⁻ ≈ –0.16 V at pH 7) (Drechsel et al. 2009a; Hassan et al. 1984; Bus et al. 1974; Drechsel et al. 2009b; Bus et al. 1984; Sawyer et al. 1981). In animal systems, redox cyclers can drive oxidative stress (Fussell et al. 2011), contributing to toxicological outcomes such as neurodegeneration. Mitochondrial complex I is especially efficient at reducing redox cyclers due to its highly negative redox centers, which matches the redox requirements of these compounds better than other mitochondrial sites like complex III (Castello et al. 2007; Cocheme et al. 2008; Drechsel et al. 2009b) 

Redox cyclers are a class of exogenous or endogenous compounds that undergo cyclic one-electron reductions and subsequent reoxidations, facilitating the continuous transfer of electrons from reducing equivalents to molecular oxygen, thereby enhancing the formation of reactive oxygen species (ROS). Within the mitochondrial respiratory chain, particularly at complexes I and III, redox cyclers can substantially amplify ROS production through their molecular interactions with redox-active centers, bypassing normal electron flow and promoting aberrant oxygen reduction. This mechanistic interaction contributes to mitochondrial oxidative stress and cellular injury and is a key feature in the pharmacological activity or toxicity of various quinone-based compounds, including certain chemotherapeutics and environmental toxins. 

Fig.2. Chemical redox cycling in mitochondria. Complex I and Complex III start PQ redox cycle in bovine heart and brain mitochondria respectively, while the involvement of outer mitochondrial membrane NADH-oxidoreductase is controversial. OMM: outer mitochondrial membrane, IMM: inner mitochondrial membrane. 

Complex I and Redox Cyclers. Complex I (NADH:ubiquinone oxidoreductase) contains multiple redox centers, including flavin mononucleotide (FMN) and a series of iron–sulfur (Fe–S) clusters, which facilitate electron transfer from NADH to ubiquinone (Verkhovskaya et al. 2008; Dutton et al. 1998; Medvedev et al. 2010; Ohnishi et al. 1998). Redox-active compounds, particularly quinones such as paraquat and menadione, interact with complex I by accepting electrons from the reduced FMN or Fe–S centers. Once reduced, these compounds can rapidly donate their acquired electrons to molecular oxygen, generating superoxide (O₂•⁻) in a catalytic cycle independent of normal ETC function. This redox cycling occurs predominantly at the FMN site of complex I, which is accessible to hydrophilic redox cyclers (Cocheme et al. 2009). These molecules effectively compete with endogenous electron acceptors for reduced FMN, diverting electrons toward oxygen rather than to ubiquinone. As a result, the presence of redox cyclers increases the rate of one-electron oxygen reduction, resulting in elevated superoxide formation. Importantly, this mechanism does not require electron flow through the entire ETC, and ROS production can proceed even when downstream complexes are inhibited or collapsed by membrane depolarization (Cocheme et al. 2008; Cocheme et al. 2009). 

Complex III and Redox Cyclers. Complex III (ubiquinol:cytochrome c oxidoreductase) is another major site of ROS production under the influence of redox-active molecules. The Q-cycle mechanism of complex III involves the oxidation of ubiquinol at the outer Qo site and the reduction of ubiquinone at the inner Qi site. During this process, a semiquinone intermediate is formed at the Qo site, which can react with oxygen to produce superoxide (Zhang et al. 2007). Lipophilic redox cyclers, such as some synthetic naphthoquinones and anthraquinones, can penetrate the inner mitochondrial membrane and localize at or near the ubiquinone-binding sites. These molecules can either act as artificial substrates for the Qo site or influence the redox state of ubiquinone intermediates by enhancing semiquinone stabilization (Castello et al. 2007). This interaction increases the lifetime and reactivity of the semiquinone intermediate, facilitating superoxide formation. Moreover, redox cyclers may promote the formation of aberrant redox couples that participate in futile cycling, further driving ROS generation. In addition to directly interacting with the redox centers of complex III, redox cyclers may alter the intramembrane redox balance, leading to an over-reduction of the ubiquinone pool. This condition is conducive to reverse electron transport (RET) at complex I, creating a feed-forward loop of ROS amplification involving both complexes. 

Redox cycler detection by EPR:

Electron paramagnetic resonance (EPR) spectroscopy provides a highly sensitive and specific method for detecting paramagnetic intermediates such as semiquinone radicals, thereby offering a direct probe into redox cycling events at the mitochondrial level. Through the application of EPR spectroscopy under controlled oxygen and substrate conditions, it is possible to monitor the formation and stability of these intermediates in situ. EPR spectra typically reveal distinct signal patterns corresponding to different redox states and radical species, allowing for the differentiation between catalytic redox cycling and simple redox reactions. Spin trapping agents may be employed to enhance detection of transient radical species, and temperature control enables the stabilization of labile intermediates.

In the presence of molecular oxygen, the interaction of a redox cycler with O₂ leads to the formation of superoxide (^·O₂^-), and concomitant regeneration of the redox cycler. Measurements have therefore be conducted under O₂-free conditions. The redox cycler is characterized by the delocalization of the unpaired electron across the conjugated ring system, allowing detection of a distinct EPR spectrum. Reduction can be achieved with sodium dithionite (Drechsel et al. 2009a; Cocheme et al. 2009; Minakata et al. 1988)

Redox cycler detection by spectrophotometry

Electron paramagnetic resonance (EPR) spectroscopy provides a highly sensitive and specific method for detecting paramagnetic intermediates such as semiquinone radicals, thereby offering a direct probe into redox cycling events at the mitochondrial level (Cocheme et al. 2009). Through the application of EPR spectroscopy under controlled oxygen and substrate conditions, it is possible to monitor the formation and stability of these intermediates in situ. EPR spectra typically reveal distinct signal patterns corresponding to different redox states and radical species, allowing for the differentiation between catalytic redox cycling and simple redox reactions. Spin trapping agents may be employed to enhance detection of transient radical species, and temperature control enables the stabilization of labile intermediates. 

In the presence of molecular oxygen, the interaction of a redox cycler with O2 leads to the formation of superoxide (·O2-), and concomitant regeneration of the redox cycler (Hassan et al. 1984; Bus et al. 1974; Bus et al. 1984; Drechsel et al. 2009a). Measurements have therefore be conducted under O2-free conditions. The redox cycler is characterized by the delocalization of the unpaired electron across the conjugated ring system, allowing detection of a distinct EPR spectrum. Reduction can be achieved with sodium dithionite (Drechsel et al. 2009a; Cocheme et al. 2009; Minakata et al. 1988). 

An example of an EPR spectrum of the PQ•+ radical is shown in Figure 3. 

Fig. 3. Detection and quantification of the PQþ radical by EPR spectroscopy. (A) Typical EPR spectrum of the PQþ radical (100 mM; trace (a)) generated in vitro by reduction of PQ2þ with a twofold excess of sodium dithionite. EPR signal of the SO 2 radical present in the dithionite solution (10 mM; trace (b)). Modified after Cocheme and Murphy, 2009 Methods in Enzymology, Copyright (2009)) Cocheme and Murphy, 2009 

Redox cycler detection by spectrophotometry 

The detection of radical formation within redox cyclers by spectrophotometry relies on the ability to monitor changes in absorbance associated with the generation and stabilization of specific radical intermediates, most commonly semiquinone radicals. These radicals are formed during one-electron redox processes when the redox-active compound cycles between fully oxidized and fully reduced states, and they often exhibit characteristic UV-visible absorption spectra that can be exploited for detection and quantification. 

Redox cyclers such as quinones (e.g., menadione, duroquinone) or nitroaromatic compounds can form semiquinone radicals upon single-electron reduction, typically mediated by mitochondrial or cytosolic reductases. The semiquinone radical is a transient species that displays distinct absorbance bands, often in the visible region (e.g., 400–600 nm), depending on the chemical structure of the redox cycler. For instance, the semiquinone form of menadione exhibits an absorption maximum around 430 nm. By recording the absorption spectrum during redox cycling under anaerobic or low-oxygen conditions (to prevent rapid reoxidation), the formation of the radical intermediate can be detected in real time. 

The paraquat (PQ•+) radical has an absorption spectrum with a maximum at 603 nm (e = 13.600 M-1cm-1). Experiments can be conducted with isolated mitochondria in an O2-free system (samples purged with nitrogen or argon). An increase in the absorption at 603 nm indicates an elevated formation of PQ•+. Mitochondrial preparations exhibit absorptions in the range of 500 nm – 700 nm. To confirm that the absorbance at 603 nm is based on PQ•+ signal, the samples are scanned from 500-700 nm under anaerobic conditions and subsequently under aerobic conditions. The presence of O2 converts PQ•+ back to PQ2+. The difference spectrum eliminates the influence of mitochondrial absorption Cocheme et al. 2009; Mayhew et al. 1978). 

Spectrophotometric detection of these intermediates often requires strict control of experimental conditions, such as oxygen tension, pH, and the presence of reducing agents (e.g., NADH, dithionite) or enzymatic systems (e.g., NADPH:cytochrome P450 reductase). These factors influence the equilibrium between fully oxidized, semiquinone, and fully reduced forms of the compound. By plotting the absorbance as a function of time or reductant concentration, it is possible to infer the kinetics of radical formation and decay, as well as the redox potential and stability of the intermediate species. 

In certain cases, differential spectrophotometry (i.e., subtraction of baseline spectra) can enhance the detection of weak or overlapping signals. Furthermore, kinetic analysis of spectral changes can reveal mechanistic aspects of redox cycling, such as disproportionation reactions or the role of molecular oxygen in promoting radical turnover (Thor et al. 1982) 

Isolated mitochondria, cultured cells and whole organisms like yeast, worms, flies, rodents and plants generate O2° in the presence of redox chemicals like Paraquat mostly increasing mitochondrial  oxidative damage (Mason, 1990; Vanfleteren, 1993, Sturz and Culotta, 2002; Van Remmen et al.,2004; Bonilla et al., 2006). Mitochondria as a major site of mitochondrial superoxide production by PQ are supported in rodents, flies and yeast. Thus, mice heterozygous for MnSOD (the isoform of superoxide dismutase locate in the mitochondrial matrix) (Van Remmen et al., 2004) and flies silenced for MnSOD (Kirby et al., 2002) show greater sensitivity to PQ than the control; flies overexpressing catalase in mitochondria are resistant to PQ, whereas enhancement of cytosolic catalase was not protective (Mockett et al., 2003); human peroxiredoxin 5 in mitochondria protects yeast more efficiently against PQ than expression in the cytosol (Tien Nguten-nhu et al., 2003). Complex I has a highly conserved subunit composition in eukaryotes (Cardol, 2011). Fourteen subunits are considered to be the minimal structural requirement for physiological functionality of the enzyme. These units are well conserved between bacterial (E. coli), human (H. sapiens), and bovine (B. Taurus) (Ferguson, 1994; Vogel et al., 2007). However, the complete structure of Complex I is reported to contain between 40 to 46 subunits and the number of subunits differs, depending on the species (Gabaldon 2005; Choi et al., 2008). Complex I is well-conserved across species, from lower organism to mammals. In vertebrates it consists of at least 46 subunits (Hassinen, 2007), including human in which 45 subunits were found (Vogel et al., 2007). Moreover, enzymatic and immunochemical evidence indicate a high degree of similarity between mammalian and fungal counterparts (Lummen, 1998). Mammalian complex I structure (Vogel et al., 2007) and activity is characterised in detail, referring to different human organs including brain. There is also substantial amount of studies performed on human muscles, brain, liver as well as bovine heart (Okun et al., 1999). Yeasts lack Complex I but reduce PQ in dependence on NADPH by intramitochondrial NADPH dehydrogenases (Cocheme and Murphy, 2008). Cytochrome bc1 complexes (Complex III) are found in the plasma membranes of photosynthetic and respiring bacteria and in the inner mitochondrial membrane of all eukaryotic cells (Trumpower, 1990). In all of these species the bc1 complex contain three electron transfer proteins and transfer electrons from a low-potential quinol to a higher-potential c-type cytochrome (Trumpower, 1990). The number of subunits in the bc1 varies between 3 catalytic subunits in some bacteria and 11 subunits in the mitochondrial bc1 (Trumpower, 1990). 

Evidence for Chemical Initiation of this Molecular Initiating Event (MIE) 

The most studied examples of chemicals that accept an electron from the mitochondrial respiratory chain and undergo redox cycling in dopaminergic neurons are the three bipyridyl herbicides paraquat, diquat and benzyl viologen. Substantial evidence has accumulated in the existing literature suggesting a role for these chemical, and paraquat in particular, and this AOP. Therefore, the redox cycler parquat will be discussed in the context of all KEs identified in this AOP. 

Paraquat as a mitochondrial electron acceptor 

The cellular toxicity of PQ is essentially due to its redox cycling abilities. Mitochondria are a major source of PQ-induced ROS production in brain (Castello et al., 2007; Figure 2). The early involvement of mitochondria in PQ-induced oxidative stress has been also demonstrated in whole cells overexpressing reduction-oxidation sensitive fluorescent proteins targeted to mitochondria or the cytosol (Rodriguez-Rocha, 2013; Filograna et al., 2016). Both Complex I (Cocheme and Murphy, 2008) and Complex III (Castello et al., 2007; Drechsel and Patel, 2009) have been involved in PQ radicalisation. In Castello et al. (2007), the redox cycle-initiating electrons are accepted from complex III and to a minor part by complex I as inhibition of complex I by rotenone only partially inhibited PQ-induced ROS formation in isolated brain mitochondria or rat midbrain cultures, while PQ-induced ROS formation in these systems was completely blocked after inhibition of complex III by using antimycin A (Castello et al., 2007; Drechsel and Patel, 2009). That complex I is not the major source of electrons triggering PQ toxicity is supported by Choi et al., (2008) who demonstrated that silencing a major component of complex I abolishing its activity does not protect against PQ-dependent dopaminergic cell death. On the other hand, Cocheme and Murphy (2008) demonstrated that PQ accumulates into yeast and bovine heart mitochondrial matrix in dependence on mitochondrial membrane potential. In heart mitochondria, PQ is then reduced mainly by Complex I forming the radical which rapidly react with O2 to give O2. The Authors explain this discrepancy with differences existing between brain and heart mitochondria (Cocheme and Murphy, 2008; Drechsel and Patel, 2009). The involvement of mitochondrial enzymes other than Complex I and III (VDAC and Cytb5, located at the external mitochondrial membrane) remains controvertial (Shimada et al., 2009; Nikiforova et al., 2014) and potentially excluded by the recent observation that the main site of PQ reduction is inside mitochondria (Nikiforova et al., 2014). 

General characteristics of other mitochondrial redox cyclers 

Other mitochondrial redox cyclers include two other bipyridyl herbicides, diquat and benzyl viologen (Figure 4 A and B). These share common structural features with paraquat (Figure A.27C): all compounds are composed of two aromatic rings containing a positively charged nitrogen and are thus good electron acceptors and redox cyclers (Drechsel and Patel, 2009; Sandy et al., 1986). 

Fig. 4. Molecular structures of: (A) diquat, (B) benzyl viologen, (C) paraquat (Copyright Drechsel and Patel, 2009, Fig. 1. Published by Oxford University Press on behalf of the Society of Toxicology) 

Quinones (i.e. menadione, Adriamycin) and nitroaromatic compounds (i.e. nitrofurantoin) also radicalise following one electron reduction by mitochondrial reductases (complex I and III and external mitochondria NADH-oxidoreductase) establishing a redox cycle (Frei et al., 1986; Nikiforova et al., 2014). Intriguingly, free cytosolic dopamine spontaneously oxidises to produce different quinones like dopamine-o-quinone and aminochrome. Aminochrome can undergo a one-electron reduction by NAD (P)H flavoproteins generating a leukoaminochrome-o-semiquinone radical and giving rise to redox cycle with production of superoxide anion (Zoccarato et al., 2005; Munoz et al., 2012). 

Fig. 5 One electron reduction of aminochrome (adapted from Mu~ noz et al., 2012, fig. 6, CC-BY) Aminochrome has been recently suggested to play a role in the death of dopaminergic neurons containing neuromelanin triggering oxidative stress/mitochondrial dysfunction, the formation of a-synuclein and impaired protein degradation (Sandy et al., 1986; Drechsel and Patel, 2009). 

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