The nuanced role of mitochondrial ROS in modulating aging and aging hallmarks

Abstract

Aging is characterized by a progressive decline in cellular integrity and function, making it a major risk factor for numerous disease pathologies. Mitochondrial dysfunction and oxidative stress have long been recognized as contributors to the aging phenotype. The loss of mitochondrial function and the overproduction of reactive oxygen species (ROS) are linked to many hallmarks of aging and are associated with a wide range of diseases; however, their role in the aging process is nuanced. Mitochondria produce ROS as harmful respiratory byproducts, but ROS can also act as a signaling molecule with emerging functions linked to variables such as location, timing, and quantity. Similarly, mitochondrial dysfunction is often broadly categorized, overlooking its multifaceted nature and diverse contributions to aging. Due to this complexity, our understanding of how mitochondrial ROS production shapes disease processes and aging hallmarks remains limited. This review aims to clarify the complex and nuanced role of mitochondrial ROS in aging by focusing on ROS production within mitochondria, especially complexes I, II and III, and exploring how these localized ROS influence various hallmarks of aging to contribute to the aging phenotype.

1. Introduction

Aging is a complex, multifactorial process defined by a gradual, time-dependent decline in cellular integrity and function, accompanied by molecular and phenotypic changes that collectively increase vulnerability to disease and death. As a major risk factor for many human pathologies, including neurodegeneration, cardiovascular disease, and cancer, the fundamental biological mechanisms of aging remain incompletely understood. Over time, a framework of cellular and molecular hallmarks of aging has been established, offering a conceptual foundation for understanding how diverse physiological alterations influence common aging phenotypes [1,2]. These hallmarks — comprising genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, disabled macroautophagy, deregulated nutrient-sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, chronic inflammation, and dysbiosis — are mechanistically interconnected rather than independent, such that disruption of one can initiates or amplifies others [2]. Each hallmark is defined by three criteria: it must manifest during physiological aging, accelerate the aging process when experimentally intensified, and slow aging or extend healthspan when experimentally ameliorated, collectively serving as both a mechanistic driver of aging and a potential entry point for therapeutic interventions [1,2].

Central to the molecular mechanisms driving aging is the dysregulation of reactive oxygen species (ROS), a chemically diverse group of oxygen-derived molecules, including superoxide (O₂^•-), hydrogen peroxide (H₂O₂), and the hydroxyl radical (^•OH), with their roles in aging being redefined over the last decades [3]. The Free Radical Theory of Aging (FRTA), first postulated by Harman in 1956, suggests that aging results from cumulative oxidative damage inflicted by ROS generated as byproducts of normal aerobic metabolism, framing aging as an intrinsic, metabolically driven process [3,4] (Fig. 1, A). This framework was extended by Harman himself and subsequently by Miquel et al. into the Mitochondrial Free Radical Theory of Aging (MFRTA), which identifies mitochondria as the primary source of endogenous ROS and proposes that the rate of mitochondrial ROS production, coupled to oxygen consumption through the respiratory chain, serves as a key determinant of lifespan across aerobic species [[5], [6], [7]] (Fig. 1, B). Although early studies in Drosophila appeared to support the oxidative damage theory, with Orr and Sohal (1994) demonstrating that simultaneous overexpression of copper-zinc superoxide dismutase (CuZnSOD) and catalase extended lifespan [8]. Later attempts to replicate this finding using single or combined antioxidant enzyme overexpression produced inconsistent results, with some studies showing a protective effect against oxidative stress [[9], [10], [11]], while other studies showed no impact on lifespan [[12], [13], [14]]. Pérez et al. demonstrated that overexpression of major antioxidant enzymes, either individually or in combination, failed to extend lifespan in mice, directly challenging the prediction that reducing oxidative damage is sufficient to promote longevity [14]. Furthermore, genetic studies in C. elegans demonstrated that mitochondrial mutations in the electron transport chain (ETC) subunits increased mitochondrial superoxide levels without affecting overall ROS levels, and paradoxically extended lifespan, contradicting that ROS decrease promotes longevity and suggesting that the relationship between mitochondrial ROS and aging is more complex [15]. Similarly, RNA interference (RNAi)-mediated knockdown of ETC subunits in Drosophila extended lifespan in a dose-dependent manner, where moderate suppression produced longevity benefits while stronger inhibition was detrimental [16]. These contradictory findings have contributed to the development of the “mitohormesis” theory, which proposes that moderate and transient elevations in mitochondrial ROS activate adaptive stress-response pathways that enhance cellular resilience and delay aging [17] (Fig. 1, C). Mechanistically, mitohormetic ROS signaling activates conserved stress-response pathways, including the p38 MAPK (PMK-1) signaling, Nrf2/SKN-1-mediated antioxidant transcription, DAF-16/FOXO nuclear translocation, and the mitochondrial unfolded protein response (UPRmt) [[17], [18], [19], [20]]. Supporting this concept, moderately increased ROS levels have been linked to enhanced longevity in C. elegans, Drosophila, and mice [16,[21], [22], [23]]. However, the mitohormetic concept has been questioned by findings indicating that specific ROS species, their site of its production within the mitochondrion, and the downstream signaling pathways they engage, rather than the magnitude of ROS elevation alone, critically determine whether the outcome are beneficial or detrimental [24]. These insights establish the need for greater mechanistic precision in characterizing mitochondrial ROS.

Fig. 1.

The nuanced role of reactive oxygen species and aging.

Schematic overview of the theoretical frameworks linking ROS to aging phenotypes. A) The Free Radical Theory of Aging; ROS generated as metabolic byproducts accumulate oxidative damage and driving aging. B) The Mitochondrial Free Radical Theory of Aging; Mitochondria as the primary ROS source, leading to accumulation of mutations, oxidative impartment, and molecular damage. A self-amplifying cycle of ROS-induced mitochondrial damage progressively increasing ROS output during aging. C) Moderate, transient ROS activates adaptive longevity pathways while excessive or chronic ROS drives pathological aging phenotypes and hallmarks. Figure was generated using Biorender.com.

ROS are now recognized as spatially and temporally regulated signaling molecules. Their biological effects are shaped not only by their concentration but also by their chemical composition, subcellular sites and duration of their production, and the targets they engage [24]. Superoxide, the primary ROS generated by complexes I, II, and III of the ETC, can undergo dismutation to form H₂O₂. As a membrane-permeant molecule, H₂O₂ can diffuse to targets outside the mitochondria or react further to hydroxyl radicals. Each transformation alters the potential range and nature of the subsequent biological effects [25,26]. The ability of complex III to release superoxide on both sites of the inner mitochondrial membrane during the Q cycle, and the role of electron flow through complex I during reverse electron transport (RET) in determining the rate and site of superoxide production have significantly advanced our understanding of mitochondrial ROS biology [27,28].

Mitochondria serve as a metabolic hub, generating the bulk of cellular energy in the form of ATP through oxidative phosphorylation while also regulating intermediate metabolism, calcium signaling, and redox homeostasis. Mitochondrial dysfunction is an established hallmark of aging, supported by evidence that biological processes dependent on mitochondrial activity progressively decline with age across species [2,29,30]. As the primary intracellular site of ROS production, mitochondria generate superoxide and hydrogen peroxide from multiple distinct sites within the ETC and associated metabolic enzymes, with complexes I, II, and III well-characterized sources. Critically, these sites vary in their sub-organellar localization, releasing ROS into the matrix, the intermembrane space, or both, in the electron donor substrates that fuel their ROS production, and the downstream targets they affect, resulting in mechanistically distinct biological outcomes when disruptions occur. This mechanistic heterogeneity, rather than the overall mitochondrial ROS output, should be the focus of aging research aimed at understanding the therapeutically targeting ROS-dependent aging processes [25,27,31].

Under physiological conditions, mitochondrial antioxidant systems, including MnSOD, glutathione peroxidase, peroxiredoxins, and thioredoxin, maintain mitochondrial redox homeostasis by neutralizing superoxide and H₂O₂²⁵. With aging, the progressive decline in antioxidant enzyme activity, mitochondrial quality control and ETC integrity disrupts this balance, leading to oxidative stress and mitochondrial dysfunction [[31], [32], [33], [34]]. Transient, moderate elevation in site-specific mitochondrial ROS, particularly during periods of metabolic stress, activate the adaptive mitohormetic pathways described above. In contrast, chronic or excessive ROS production, arising when antioxidant defenses are overwhelmed by prolonged ETC dysfunction, or age-related mitochondrial decline, drives gradual accumulation of oxidative damage that contributes to aging phenotypes [2,17,35,36]. Dysregulated mitochondrial ROS have downstream effects that influence the hallmarks of aging: oxidative DNA damage contributes to genomic instability, while ROS-dependent inhibition of epigenetic enzymes alters histone and DNA methylation patterns. Protein oxidation disrupts proteostasis and autophagic clearance, and redox modulation of AMPK and mTOR signaling impairs nutrient-sensing. Mitochondrial ROS also activate the NLRP3 inflammasome, driving chronic inflammation, and promote senescence-associated secretory phenotypes. Additionally, impaired mitochondrial function in stem cells drives exhaustion, reduced regeneration capacity, and altered intercellular communication within tissues [22,[36], [37], [38], [39]].

Rather than being determined by mitochondrial ROS as a uniform entity, these effects are dependent on several factors, including the specific ETC site of ROS production, the direction of electron flow through the generating complex, the magnitude and duration of ROS production, and the subcellular compartment into which ROS is released. Critically, Bleier et al. demonstrated that ROS generated at complex I and III modifies separate, non-overlapping sets of target proteins, providing direct evidence for generator-specific redox signaling functions. This site-specificity provides mechanistic basis for how mitochondrial ROS can function both as signaling molecules and as mediator of cellular damage but targeting distinct protein pools [40]. To advance our understanding, it is essential to recognize the nuanced nature of ROS and shift research focus from the generalized concept of ‘mitochondrial ROS’ to a more precise and detailed framework. This approach should account for the context-dependent ROS production within the ETC, and therefore the context-dependent outcome. This review focuses on ROS production at complexes I, II, and III of the ETC and systematically examines how ROS from each complex contributes to the aging phenotype and established aging hallmarks. For each complex, we first established the mechanistic basis of ROS production, including specific sites, electron donors, and conditions that drive ROS generation, before examining the evidence linking site-specific ROS to aging phenotypes across model organisms, mammalian systems, and human studies. By connecting specific ETC-derived ROS sources to mechanistically defined hallmark outcomes, this review aims to move the field beyond the broad framing of ‘mitochondrial oxidative stress’ toward site-specific, mechanistically precise understanding of how ETC-derived ROS shapes the aging process and to identify the molecular targeted most suitable for therapeutic interventions.

2. Mitochondrial ROS production sites

Mitochondrial ROS are generated at multiple sites within the organelle [25,31,41,42]. Among these, complexes I, II, and III of the ETC represent the most extensively characterized sources of superoxide and H₂O₂ and are the principal focus of this review (Fig. 2). Mitochondrial ROS are generated within the ETC when electrons leak from respiratory complexes and directly reduce molecular oxygen to form superoxide, with each site targeting specific compartments [25].

Fig. 2.

The electron transport chain (ETC) and mitochondrial ROS production

The ETC is essential for oxidative phosphorylation and consists of enzyme complexes embedded in the inner mitochondrial membrane, typically referred to as complexes I (blue), II (dark green), III (light green) and IV (light grey), along with two mobile electron carriers: ubiquinone (CoQ, purple) and cytochrome C (CytC, purple). NADH is oxidized by complex I, and FADH₂ by complex II, transferring electrons to CoQ. Electrons then flow from CoQ to complex III, CytC, and complex IV, where oxygen is reduced to form water. Complexes I, III, and IV pump protons into the intermembrane space, creating an electrochemical gradient that drives ATP synthesis via complex V (dark grey). A byproduct of the ETC activity is the generation of ROS (red stars). Forward electron flow (dark arrows), reverse electron flow (RET, red arrow). Figure was generated using Biorender.com.

At complex I, ROS are generated at two distinct sites, the flavin mononucleotide (FMN) site and the ubiquinone-binding site, during both forward electron transport (FET), in which electrons from NADH are transferred through iron-sulfur clusters to ubiquinone (CoQ), and reverse electron transport (RET), in which electrons flow backwards through complex I, driven by a highly reduced CoQ pool and elevated mitochondrial membrane potential, generating at the FMN site [25,31]. ROS generation at complex II occurs at the flavin site (II_F) and the ubiquinone-binding site and is influenced by succinate concentration [43]. During FET, complex II oxidizes succinate to fumarate, reducing FAD to FADH₂. Electrons are then transferred through iron-sulfur clusters to ubiquinone, which subsequently delivers electrons to complex III [25,31]. At complex III, ROS is generated during the Q cycle, when a semiquinone radical forms transiently at the Qo site, which can react with molecular oxygen to produce superoxide. Slower electron transfer increases semiquinone accumulation, raising the likelihood of ROS production. Complex III-derived ROS can be released into both the matrix and the intermembrane space [25,31].

Beyond the individual contributions of complexes I, II, and III, emerging evidence indicates that the physical organization of individual ETC complexes into stable, stoichiometric assemblies, known as supercomplexes (SC) or respirasomes, represents an additional layer of regulation that influences mitochondrial ROS output and may possibly contribute to aging phenotypes and aging hallmarks [[44], [45], [46], [47]]. SC are formed within the inner mitochondrial membrane, primarily incorporating complexes I, III, and IV, and are stabilized by cardiolipin, structural proteins like the MICOS complex, and assembly factors such as SCAF1 and COX7A2L [[44], [45], [46], [47]]. The most prominent SC in mammalian mitochondria is the I₁III₂IV₁ respirasome. SC are proposed to improve ETC efficiency by transporting electrons more directly between complexes, reducing electron leakage, and thereby limiting mitochondrial ROS generation. SC also help maintain the structural integrity of ETC complexes and prevent protein aggregation within the densely populated inner mitochondrial membrane. The plasticity model of the ETC proposes that ETC complexes can dynamically alternate between free and SC-associated states in response to metabolic demands, with this adaptability potentially declining with age [48]. For example, Lopez-Fabuel et al. showed that neurons and astrocytes differ in mitochondrial respiratory chain organization, with astrocytes having more free complex I and neurons assembling more complex I in SC. These structural differences are linked to variations in respiratory efficiency and ROS production, with increased ROS levels associated with the higher prevalence of free complex I [49]. Maranzana et al. demonstrated that disruption of SC in bovine heart mitochondria significantly increased superoxide production form complex I [50]. Supported by studies that show that SC destabilization increases oxidative stress in aging tissues [51]. Age-related SC disassembly has been observed in various tissues, with proteomic studies in aged rat mitochondria linking changes in OXPHOS SC architecture to increased ROS production. Interventions, such as caloric restriction or the overexpression of SC assembly factor COX7RP, have been shown to enhance SC assembly, improving mitochondrial function and promoting longevity [[52], [53], [54]]. Together, these findings suggest that age-related decline in SC assembly represents an additional mechanism through which mitochondrial aging amplifies site-specific ROS production with downstream consequences for the aging hallmarks.

Besides these three complexes, additional mitochondrial sources, including the pyruvate dehydrogenase complex, α-ketoglutarate dehydrogenase complex, and monoamine oxidases, may contribute to aging phenotypes as well but are beyond the scope of this review [25,31]. Superoxide is membrane-impermeant and acts locally at its site of production. Its dismutation, catalyzed by manganese superoxide dismutase (MnSOD) in the mitochondrial matrix or CuZnSOD in the intermembrane space, generates H₂O₂, which is membrane-permeant and can diffuse to cytosolic and nuclear compartments to engage redox-sensitive signaling targets. H₂O₂ can further react to the highly reactive hydroxyl radical, causing irreversible oxidative damage to DNA, proteins, and lipids. The progression from superoxide to H₂O₂ to hydroxyl radical highlights the role of the initial site of generation within the mitochondrion, as it determines which dismutase acts on the superoxide, the compartment, and the downstream targets that are impacted [25,26]. This context-dependent interconversion of ROS species implies that the biological effects of superoxide production at a specific ETC site can influence regions beyond its point of origin, making it challenging to attribute distinct biological outcomes to individual production sites. Despite this complexity, research increasingly links distinct sites of ETC-derived ROS production to specific biological targets and signaling outcomes, challenging this historical view of mitochondrial ROS as uniformly damaging byproducts of the ETC.

It is important to acknowledge that different ETC-derived ROS sites can influence the same aging phenotypes and hallmarks through distinct pathways, under different conditions, at different magnitudes, and at different timepoints. This does not diminish the significance of site-specific attributions, rather it highlights that the same aging hallmarks can arise from mechanistically distinct upstream signals, a distinction with direct relevance for therapeutic strategies. The following section examines how mitochondrial ROS collectively influences the hallmarks of aging, before systematically exploring site-specific ETC-derived ROS generation and its age-related effects.

3. Mitochondrial ROS influence aging hallmarks

Mitochondrial dysfunction is a central hallmark of aging, that both is amplified and drives progressive decline of other hallmarks through extensive bidirectional interactions. This positions mitochondrial dysfunction and in particular dysregulated mitochondrial ROS production as a central mechanism which molecular damage spreads across hallmark boundaries [2,32,[36], [37], [38], [39]]. Mitochondrial dysfunction encompasses a wide range of processes, reflecting the complexity of mitochondrial biology and their involvement in numerous cellular functions beyond energy production. Mitochondrial ROS are a major driver of the decline and interactions, influencing the entire range of aging hallmarks. The extent of this influence is determined by the site of ROS generation, the specific species, and the surrounding cellular context.

Among the primary hallmarks (Fig. 3, blue), mitochondrial ROS impairs genomic integrity through oxidative DNA lesions that drive mutagenesis and compromise genome stability [55]. Telomeres are highly susceptible to oxidative stress, and mitochondrial ROS-driven telomere shortening further contributes to replicative senescence and genomic instability [56]. ROS-mediated inhibition of DNA and histone-modifying enzymes disrupt epigenetic regulation, leading dysregulated gene expression [57]. Proteostasis is disrupted when mitochondrial ROS oxidize proteins, impair their proper folding, and compromise cellular quality-control mechanisms [58]. Furthermore, mitochondrial ROS can interfere with autophagic processes, such as mitophagy, reducing the cell's ability to clear damaged organelles and maintain energy homeostasis, which exacerbates mitochondrial dysfunction [59]. Mitochondrial ROS also engaged the antagonistic hallmarks of aging (Fig. 3, green). Mitochondrial ROS modulates nutrient-sensing pathways, including AMPK and mTOR, through redox-dependent alterations that disrupt the metabolic regulation essential for healthy aging [60]. Cellular senescence is driven through oxidative activation of DNA damage response pathways, resulting in irreversible cell cycle arrest, and the secretion of pro-inflammatory factors and exacerbating tissue dysfunction [61]. Mitochondrial dysfunction itself is both a driver and a result of ROS, with excessive ROS production damaging ETC components, further impairing mitochondrial function and leading to a cycle of oxidative stress and bioenergetic decline [62]. At the integrative hallmark level (Fig. 3, orange), mitochondrial ROS drives chronic inflammation through activation of inflammatory signaling cascades [63]. Stem cell exhaustion is promoted by ROS-mediated disruption of stem cell renewal and function, impairing tissue regenerative capacity [64]. ROS-induced damage to epithelial barrier integrity leads to intestinal barrier dysfunction and dysbiosis, triggering systemic inflammation and accelerating the aging process [65]. Altered intercellular communication is mediated by ROS-dependent modifications of signaling molecules, altering signaling pathways and promoting oxidative stress in neighboring cells [66].

Fig. 3.

Mitochondrial dysfunction impacts the hallmarks of aging.

Illustration of mitochondrial ROS as a central feature of mitochondrial dysfunction (center) and its widespread effects on the hallmarks of aging. ROS produced in the mitochondria can affect the primary hallmarks (blue, genomic instability, telomere attrition, epigenetic alterations, loss of proteastasis, and disabled macroautophagy. Mitochondrial ROS also impacts antagonistic hallmarks (green, deregulated nutrient-sensing, mitochondrial dysfunction, and cellular senescence). Additionally, mitochondrial ROS can affect the integrative hallmarks (orange, stem cell exhaustion, altered intercellular communication, chronic inflammation, and dysbiosis. Figure was generated using Biorender.com.

Together, these findings demonstrate that mitochondrial ROS production is an active driver of hallmark-level cellular decline throughout the aging process. However, the influence described above should not be attributed to mitochondrial ROS as a uniform entity. As the studies discussed in the following sections demonstrate, factors such as the site-specific ROS production, the direction of electron flow, and the intensity and duration of ROS production, are critical for the aging outcomes. The following sections focus on a site-specific context-dependent perspective, rather than the broad concept of mitochondrial oxidative stress, by exploring evidence that links ROS from complexes I, II, and III specifically to aging hallmarks and aging phenotypes.

4. Complex I

4.1. ROS production by complex I

Complex I, also known as NADH:ubiquinone oxidoreductase, is the largest component of the ETC, comprising 45 subunits organized into two structural arms (Fig. 4, A). The hydrophilic arm, encoded by the nuclear genome, extends into the matrix, and contains the catalytic core — including the NADH-binding site (NADH oxidation), as well as the iron-sulfur (Fe–S) clusters that facilitate electron transfer from FMN to CoQ [67]. The hydrophobic membrane arm, encoded by the mitochondrial genome, is embedded within the inner mitochondrial membrane and contains the machinery responsible for proton translocation [67,68]. NADH, generated primarily by the TCA cycle and β-oxidation of fatty acids, is oxidized at the FMN site of complex I and regenerates NAD⁺ in the mitochondrial matrix. Through the oxidation of NADH, two electrons are transferred and CoQ is reduced to ubiquinol. This forward electron transfer is coupled with the translocation of four protons from the matrix to the intermembrane space, contributing to the mitochondrial membrane potential and ATP synthesis [68]. In addition to its bioenergetic role, complex I is a primary source of mitochondrial ROS, specifically superoxide, which can be generated at two sites: the FMN site (I_F site) and the ubiquinone-binding site (I_Q) [[68], [69], [70], [71]]. Superoxide is released predominantly into the matrix, where it is dismutated to H₂O₂ by MnSOD or activates downstream redox-sensitive signaling targets [72]. Complex I undergoes structural and conformational changes, between active and deactive states, to regulate ROS production and adjust ETC activity [73]. Dysfunctional complex I is associated with reduced catalytic activity and increased ROS production, with the specific pathological consequences being heavily context-dependent [25,69].

Fig. 4.

Structure and ROS generation sites of complex I during forward and reverse electron transport.

A) Ribbon representation of the human complex I crystal structure illustrating the four subunits and key factors. The hydrophobic arm spans the inner mitochondrial membrane and the hydrophilic arm extends into the matrix, which contains the flavin mononucleotide cofactor (FMN, yellow), the primary ROS -generating site, a chain of iron-sulfur (Fe–S) clusters (magenta) that form the electron relay between FMN and CoQ, and the ubiquinone-binding site (I_Q, cyan) a secondary ROS-generating site. B) Forward electron transport (FET). During normal respiration, NADH donates electrons at the FMN site, which are transferred through the Fe–S cluster chain to reduce CoQ at the I_Q site. Four protons are simultaneously translocated from the matrix into the intermembrane space contributing to the mitochondrial membrane potential. Under FET conditions, mitochondrial membrane potential and CoQ pool reduction are low, resulting in minimal ROS production at the I_F site. C) Reverse electron transport (RET). When the CoQ ppol is highly reduced and mitochondrial membrane potential is elevated, electrons flow backwards through complex I, where they react with molecular oxygen to generate a burst of ROS. Figure was generated using Biorender.com and ChimeraX, PDB structure 5XTD.

Complex I generates ROS during both FET and RET, two mechanistically distinct approaches of ROS production with different physiological and pathological consequences. During FET, electrons from NADH are transferred through iron-sulfur clusters to CoQ and superoxide is generated when electrons leak to molecular oxygen, mainly at the FMN site under conditions of high NADH/NAD⁺ ratio or when downstream electron transport is disrupted. Under normal physiological conditions FET-associated complex I-derived ROS production is low, however, under pathological conditions when downstream flow is impaired, e.g. during complex I inhibition or dysfunction [25,71,72,74] (Fig. 4, B). RET occurs when electrons flow backwards through complex I, from a highly reduced CoQ pool through the iron-sulfur clusters to the FMN site, where superoxide is generated upon reaction with molecular oxygen. RET has been recognized as the primary mechanism for maximal complex I-derived ROS production, occurring when the CoQ pool is highly reduced and the mitochondrial membrane potential is high, creating the thermodynamic conditions to drive electrons in reverse to the ROS producing site within complex I [28,75] (Fig. 4, C). A key physiological driver of RET is succinate accumulation: during ischemia, succinate builds to supraphysiological concentrations and its rapid reoxidation via complex II upon reperfusion reduces the CoQ pool, driving reverse electron flow through complex I and producing a burst of matrix-directed superoxide, that contributes to reperfusion-associated oxidative stress [25,28,76].

Critically, rotenone, a highly lipophilic neurotoxicant derived from plants that crosses the blood-brain barrier and accumulates in subcellular organelles, inhibits complex I at the ubiquinone-binding site and blocks RET, allowing for discrimination between ROS produced during forward versus reverse electron transfer [[77], [78], [79]]. However, rotenone simultaneously decreases electron transfer to complex III, complicating efforts to isolate the effects of complex I ROS from broader metabolic responses. To address this limitation site-specific inhibitors of complex I I_Q-site electron leak (S1QEL1.1 and related compounds) were developed to prevent electron leak from the I_Q-site and reverse electron transfer with limited effects on forward electron transfer and enabling more precise pharmacological dissection oof site-specific complex I ROS contributions [70]. The physiological and pathological consequences of complex I RET-derived ROS are profoundly context-dependent. RET-derived ROS has been implicated in innate immune activation [80], tuberculosis [81], and ischemia-reperfusion injury [76,79,82]. Demonstrating the complexity and dual nature of RET-derived ROS, its loss impairs diastolic function, exercise capacity [83], and impairs host response to bacterial infections [84]. Beyond RET-derived ROS, complex I FET-derived ROS, as well as inhibitors that enhance ROS production, have been linked to aging phenotypes, multiple cancers, cardiovascular disease, drug induced liver injury, and diabetes [42].

Due to the functional diversity, understanding the precise contributions of complex I-derived ROS to aging has proven challenging. Prior studies have proposed two primary mechanisms through which complex I dysfunction contributes to aging: increased site-specific ROS production and alterations in the cellular NAD⁺/NADH ratio resulting from impaired electron transfer [85,86]. The following section examines the evidence linking these mechanisms to specific aging phenotypes and hallmarks across model organisms, mammalian systems, and human studies.

4.1.1. Complex I-derived ROS and its impact on aging and aging hallmarks

The initial evidence suggesting that a reduction of complex I subunits could increase lifespan was based on genome-wide RNAi screening studies in C. elegans [87,88]. These studies demonstrated that under certain conditions, C. elegans lived significantly longer, but exhibited reduced size, decreased activity and prolonged developmental time. These findings revealed that the relationship between reduced ETC complex function and increased longevity is nuanced and context-dependent [87,88]. Building on these findings, studies utilizing genetic models have provided deeper insights into how specific genetic mutations influence aging processes and stress resistance. For instance, C. elegans strains with mutations in mitochondrial genes, collectively known as mit mutants, exhibit distinct longevity phenotypes and varying mitochondrial stress resistance. While most of these mutations occurring in nuclear-encoded subunits of the ETC, the molecular mechanisms underlying these differences are under investigation [87,[89], [90], [91], [92], [93], [94], [95]]. It is hypothesized that variations in ROS signaling, damage, and stress responses contribute to the observed lifespan effects. A U-shaped relationship between ROS levels and lifespan has been proposed in these mutants, where moderate increases in ROS levels can extend lifespan, but excessive ROS levels may have detrimental effects, reducing lifespan beyond an optimal threshold [23,90,[96], [97], [98]].

Investigations in model organisms have tied complex I function and ROS with both lifespan shortening and extension. In C. elegans much work has focused on the gas-1(fc21) mutant, which carries a missense mutation in the 49 kDa subunit of complex I, and exhibits a shortened lifespan, a decrease in complex I activity, lower offspring rates, and hypersensitivity to volatile anesthetics [90]. This mutation results in a reduced maximal rate of oxidative phosphorylation through complex I, accompanied by an elevated rate of complex II-dependent activity, suggesting a compensatory shift in electron transport toward complex II upon complex I dysfunction [99]. Additionally, increased oxidative damage to mitochondrial proteins was observed, which was rescued by suppressors of gas-1 that decrease oxidative damage in mitochondria [90]. Kayser et al. demonstrated that aging in C. elegans is associated with the accumulation of oxidative damage, while sensitivity to volatile anesthetics is linked to both complex I function and oxidative damage. Analysis of mitochondrial function mutants further suggested that lifespan correlates more closely with the accumulation of oxidative damage in mitochondrial proteins than with alterations in oxidative phosphorylation capacity, suggesting that oxidative protein damage may be a more proximal determinant of lifespan than bioenergetic decline in these mutants [90].

McCormack et al. investigated whether pharmacological targeting of sirtuin and PPAR signaling pathways, given their critical roles in mediating mitochondrial physiology and the cellular response to metabolic stress, could alter observed gas-1 phenotypes. Resveratrol (a sirtuin activator) and nicotinic acid (an indirect NAD⁺ precursor) rescued the lifespan of gas-1(fc21) mutants, regardless of treatment timing, while rosiglitazone (a PPARy agonist) rescued lifespan when administered in young adulthood. Nicotinic acid reduced mitochondrial oxidant burden, whereas resveratrol unexpectedly increased ROS production. Despite this difference, both treatments produced equivalent lifespan benefits, demonstrating that the shortened lifespan was rescued through mechanisms that dissociate lifespan extension from mitochondrial oxidant burden, suggesting that restoring NAD⁺/NADH balance and improving mitochondrial membrane potential may be more critical determinates of lifespan in this context than the reduction of oxidative stress [100]. Rosiglitazone had minimal impact on oxidant burden, and fenofibrate (a PPARα agonist) modestly increased ROS without rescuing lifespan. These findings highlight a critical dissociation between ROS levels and lifespan in gas-1 mutants, suggesting that bioenergetic and redox signaling parameters interact in a complex, context-dependent manner to determine longevity outcomes [100]. Nuo-6, another C. elegans mutant for complex I, exhibits elevated superoxide levels despite overall ROS levels remaining unchanged and oxidative stress being low. Using an adapted flow cytometry technique, researchers directly measured ROS levels in isolated worm mitochondria, confirming increased superoxide production in nuo-6 mutants. This increase in superoxide was shown to be both necessary and sufficient for the lifespan extension, as demonstrated by abolition upon treatment with antioxidants (NAC and vitamin C) and its mimicry by mild treatment with the prooxidant paraquat. These findings suggest that superoxide acts as a redox signaling molecule, triggering protective cellular responses that mitigate aging. The study proposes a model in which molecular damage during aging induces a protective superoxide response, offering insight into the correlation between complex I-derived ROS and aging phenotypes [15].

Apart from complex I mutants, administration of phloretin, which inhibits complex I subunits NDUFS1 and NDUFS6, increased lifespan and fitness of C. elegans. Under oxidative stress, the survival was enhanced in an inverted U-shaped dose-dependent manner, consistent with mitohormesis in which low/moderate ROS levels act as adaptive signals while high ROS levels become damaging [17]. Its lifespan-extending effects were mediated by increased ROS through complex I inhibition, which activated the stress-responsive pathways p38 MAPK/PMK-1, NRF-2/SKN-1 and FOXO/DAF-16, collectively reinforcing stress resistance. Additionally, complex I inhibition reduced ATP levels, activating the energy sensors AMPK/AAK-2 and SIRT1/SIR-2.1 as downstream mediators of phloretin's lifespan-extending effects, thereby connecting complex I-derived ROS, bioenergetic stress and conserved signaling pathway associated with longevity [19].

Conflicting reports have emerged in Drosophila models regarding the relationship between complex I function, ROS, and lifespan. Copeland et al. demonstrated that RNAi knockdown of mitochondrial respiratory complex genes (I, III, IV, and V) extends lifespan in Drosophila. Knockdown of two complex I subunit genes (CG9172 and CG9762) throughout development and adulthood increased the flies’ mean lifespan. Targeted RNAi of these complex I genes in adult tissues or neurons alone was sufficient to extend lifespan, indicating that the effects are independent of developmental knockdown and identifying neurons as a key tissue mediating the longevity response. RNAi of complex I and III also conferred resistance to the superoxide-generating agent paraquat, while knockdown of complex IV or V subunits did not, suggesting a site-specific oxidative stress resistance mechanism linked to the predominant roles of complexes I and III as the primary source of superoxide generation [16]. Similar findings in C. elegans showed no consistent correlation between lifespan extension and reduced oxidative damage in ETC mutants, suggesting that lifespan extension through ETC alterations involves mechanisms other than reduction of ROS [16,88,89]. However, conflicting evidence exists in regard of the lifespan outcome and the timing of required intervention. One study generated RNAi knockdown fly strains targeting complex I subunits NDUFS4 and NDUFS7 and reported a decreased lifespan compared to controls [101]. Consistently, depletion of complex I subunits ND-18 (Drosophila ortholog of Ndufs4) and ND-75 (Drosophila ortholog of Ndufs1) via RNAi reduced lifespan and increases stress sensitivity only when initiated during development, while depletion restricted to adulthood only is insufficient to reduce lifespan [102]. Together, these findings indicate that the specific complex I subunit targeted and the timing of depletion are key factors influencing longevity outcomes. This could reflect subunit-specific roles in electron transport, generation of ROS, and the structural integrity of complex I assembly [16,101,102]. The site of mitochondrial ROS generation determines the effects, with increase ROS via RET at complex I delaying aging and age-related diseases in flies. Under basal conditions, aged flies exhibited elevated ROS levels, reduced complex I-linked respiration, and decreased enzymatic activity of complexes I and III [103]. Expression of the alternative NADH dehydrogenase NDI1, which bypasses canonical complex I electron flow, was previously shown to reduce ROS in some contexts [104], however, it unexpectedly increased ROS in fly brains by over-reducing the CoQ pool, thereby driving RET at complex I¹⁰³. This NDI1-mediated ROS increase, generated specifically though RET, improved mitochondrial function and extended lifespan in models of mitochondrial dysfunction, suggesting that targeted ROS production at complex I can promote health and longevity [103]. However, this lifespan extension was abolished when electron flow declined during aging [105], consistent with the dependence of RET on a highly reduced CoQ pool and a high membrane potential, both of which decline with age [76,105]. Further evidence for the adaptive and nuanced role of complex I-derived ROS comes from studies of the acute stress response. Under heat stress, ROS transiently increased in the fly brain via RET at complex I, with levels returning to baseline within hours, indicating a regulated, stimulus-specific stress signaling response rather than overall oxidative damage. Disruption of the ROS-RET signal impairs the stress response, suppresses pro-survival gene activation, and reduces lifespan, demonstrating complex I-derived ROS constitutes an essential adaptive signal for stress resistance and longevity [106].

In C. elegans, knockdown of complex I subunits during development extends lifespan, while depletion in adulthood has no effect [20]. However, the outcomes of complex I disruption are highly nuanced, varying not only between species, but also within a single species depending on the specific subunit targeted, the timing of disruption, and the extent of complex I depletion. Furthermore, studies on complex I-derived ROS reveal its dual role in either extending or reducing lifespan, depending on the precise nature of the disruption and its cellular and physiological context. These findings highlight the intricate interplay between complex I function, ROS production, and aging, and underscore the need for further research to understand how complex I-derived ROS specifically influences aging phenotypes and hallmarks.

One of the well-characterized aging hallmarks is genomic instability, appearing as oxidative damage to both mitochondrial and nuclear DNA [2]. Complex I–derived ROS has been repeatedly implicated in genomic damage and transcriptional dysregulation. Mitochondrial DNA damage induced by complex I-derived ROS is widely documented [[107], [108], [109], [110]]. Caloric restriction (CR) has been shown to reduce mitochondrial ROS production and oxidative DNA damage, primarily at complex I, in rat organs, representing one mechanistic basis by which CR slows aging-associated molecular decline. In Wistar rats, seven weeks of protein restriction resulted in a 30-40% reduction in mitochondrial ROS production and oxidative damage to nuclear and mitochondrial DNA in the liver, specifically at complex I. These changes occurred without altering mitochondrial oxygen consumption and were linked to a decrease in the proportion of electron leak [111,112]. Sanz et al. investigated the impact of late-life CR on oxidative stress in the brain, a key organ for aging. Wistar rats subjected to 40% CR initiated at 24 months of age for one year showed a 24% reduction in H₂O₂ production and a 23% decrease in mitochondrial DNA oxidative damage, with levels falling below those of both old and young ad libitum-fed animals. CR also reversed age-related oxidative damage to nuclear DNA, restoring it to levels observed in young controls. The reduction in ROS was localized to complex I, occurring without changes in mitochondrial oxygen consumption, and was linked to improved efficiency in reducing electron leak at complex I. These findings support the idea that CR reduces complex I-derived ROS and oxidative stress in the brain, even when initiated late in life, supporting its role in slowing the aging process during the final stages of the lifespan [112]. Similarly, methionine restriction (MetR) in Wistar rats reduced mitochondrial ROS production, oxidative damage to mitochondrial DNA and proteins, membrane unsaturation, and markers of protein oxidation in heart and liver mitochondria. MetR also decreased the protein levels of complexes I and IV, with decreased ROS at complexes I and III in the liver and at complex I in the heart. This was attributed to improved efficiency of the respiratory chain in minimizing electron leak to oxygen [113]. An additional study subjected Wistar rats to 40% MetR for seven weeks, revealing decreased mitochondrial ROS production at complex I during FET, reduced oxidative damage to mitochondrial DNA and proteins, and a lower degree of genomic DNA methylation. Notably, no significant changes were observed in mitochondrial oxygen consumption, the protein levels of complexes I-IV, or the mitochondrial apoptosis-inducing factor [114]. Together these findings suggest that both protein and methionine restriction reduce complex I-derived ROS production by improving the coupling efficiency of electron transfer at complex I and thereby reducing oxidative damage to mitochondrial and nuclear DNA [111,113,114]. This highlights dietary restriction-mediated optimization of complex I as a mechanistic contributor to the longevity effects of CR and MetR.

Beyond dietary modulation of complex I-derived ROS, pharmacological disruption of complex I function provides complementary mechanistic evidence for the role of complex I-derived ROS in genomic damage and neurodegeneration. Exposure to the complex I inhibitor rotenone in C6 cells, an astrocyte-like cell line derived from rat glioma, decreased cell viability, increased ROS production, and caused oxidative DNA damage. Rotenone also elevated the glial fibrillary acidic protein (GFAP), an astrocyte-specific marker, and caspase-3 expression, indicating glial activation and apoptosis. The observed toxicity was linked to complex I dysfunction, which increased ROS generation, resulting in oxidative damage to cellular components [77]. These findings are particularly relevant to aging, as mitochondrial dysfunction, oxidative stress, and glial activation are key contributors to age-related neurodegeneration and cellular senescence [7,36,77,115]. Co-treatment with the antioxidant melatonin significantly suppressed rotenone-induced effects, including ROS production, DNA damage, and caspase-3 expression, demonstrating its protective role against ROS-mediated apoptosis and highlighting the potential of targeting complex I-derived ROS to counteract neurotoxic effects and support brain health [77]. Similar evidence linking complex I dysfunction to nuclear DNA damage and genomic instability comes from studies of cybrid cell models of Parkinson's disease (PD). Using PD63, a cybrid cell line harboring mitochondrial DNA from a sporadic PD patient containing a missense mutation in the complex I ND5 subunit, researchers observed increased ROS levels, chronic nuclear DNA damage, and activation of cellular DNA damage responses upon differentiation into dopaminergic neuron-like cells [116].

A study by Winstanley et al. investigated how mitochondrial function impacts telomere elongation during preimplantation embryogenesis. Female mice exposed to rotenone produced offspring with shorter telomeres in the kidney and heart. Restricting rotenone exposure to oocytes prior to fertilization resulted in elevated ROS levels, oxidative DNA lesions, and disrupted mitochondrial membrane potential in zygotes, impairing pronuclear reprogramming and the establishment of specific epigenetic marks during early embryonic development [56]. While the rotenone concentration affects both complex I and complex II, making it difficult to attribute ROS production to a specific site, these findings demonstrate that increased ROS levels contribute to nuclear DNA damage, telomere shortening, and an increased risk of age-related diseases in offspring. Telomere shortening is an established hallmark of aging, and its induction by ROS during embryogenesis raises interest in early-life exposures that accelerate aging hallmarks and show the importance of mitochondrial health in oocytes for proper embryonic development and telomere maintenance [2,56].

Besides direct oxidative damage to DNA, complex I-derived ROS has also been implicated in epigenetic dysregulation, a mechanistically distinct but related pathway through which ROS influences nuclear gene expression and cellular aging. Epigenetic alterations represent a further molecular route through which complex I-derived ROS influences cellular aging. Rotenone exposure has been shown to induce epigenetic modifications to PD-relevant genes in HEK293 cells [117] and mouse brain organoids [118,119]. While these studies recognize rotenone as complex I inhibitor, they also suggest that rotenone may contribute to neurodegeneration through mechanisms independent of complex I inhibition. Importantly, these studies did not measure ROS production, leaving the precise contribution of oxidative stress in these epigenetic changes unclear. Targeted induction of superoxide using mitochondria-localized paraquat (MitoPQ), which selectively mimics complex I ROS production [120], has been shown to alter mitochondrial function and metabolic activity [121]. The generated superoxide mediates epigenetic modifications of histones through metabolic rewiring that increases acetyl-CoA availability, leading to changes in histone acetylation in primary neonatal human epidermal keratinocytes (HEKn) [121], thereby establishing a direct mechanistic link between site-specific complex I-derived ROS and chromatin-level regulation of gene expression [121]. Furthermore, Baeken et al. utilized the complex I inhibitor MMP⁺ to evaluate the role of complex I-derived ROS in epigenetic regulation. They concluded that mitochondria employ complex I-derived ROS as a redox signal to communicate impending, but not severe, mitochondrial distress to the nucleus, a signal in which the level of complex I ROS production determines whether the nuclear response is adaptive reprogramming or an induction of autophagic degradation [57]. This signal regulates epigenetic and transcriptional responses to mitochondrial stress in human neuronal LUHMES cells, suggesting a broader, ongoing regulatory function of complex I-derived ROS through the selective upregulation of mito-metabolic genes via chromatin reorganization [57].

One of the most rapidly expanding areas of research implicates complex I–derived ROS in immune activation and chronic inflammation. Several studies demonstrate that complex I-derived ROS contributes to sustained microglial activation, promoting chronic neuroinflammation [80,84,122]. Upon stimulation with lipopolysaccharide (LPS), macrophages shift from oxidative phosphorylation to glycolysis for ATP production, while increasing succinate levels. Succinate oxidation via complex II combined with an elevated mitochondrial membrane potential, drives RET ROS production at complex I and promotes a pro-inflammatory state through stabilization of HIF-1α and subsequent IL-1β transcription [84]. Blocking ROS production, via rotenone, mitochondrial uncoupling, or expression of the alternative oxidase (AOX) inhibits this inflammatory phenotype, indicating that mitochondria are repurposed from ATP synthesis to ROS production during macrophage activation. Succinate thus emerges as a critical metabolic regulator of the pro-inflammatory response, while suppressing anti-inflammatory gene expression [84]. This pro-inflammatory ROS signal is sensitive to interventions that disrupt RET, including mitochondrial uncoupling, inhibition of succinate oxidation, oxidation of the CoQ pool, and inhibition of complex I, collectively showing that RET at complex I is the mechanistic source of pro-inflammatory superoxide in this context [84]. Notably, rotenone reduces ROS production at complex I only when RET is occurring, paradoxically demonstrating that rotenone suppresses rather than increases ROS under RET [123]. Furthermore, metformin, which inhibits complex I, decreases IL-1β production in response to LPS [124]. TLR4 further modulates complex I activity and ROS production in inflammation [125]. Additionally, work in LPS-stimulated mice indicates that RET, and therefore the associated complex I-derived ROS, regulates IL-1β release during NLRP3 inflammasome activation in macrophages [80]. These findings firmly establish that RET at complex I drives pro-inflammatory ROS production in LPS-activated macrophages, placing complex I as a central node in the metabolic-inflammatory axis and identifying RET as a mechanistically target for modulating innate immune activation [80,84,124,125].

Notably, cell-type specificity is evident: while complex I-derived ROS primarily drives baseline ROS production in astrocytes, complex III emerges as the dominant source regulating astrocyte-mediated inflammatory responses [66]. This highlights the critical importance of identifying the precise source of ROS, as its origin and role can vary significantly across different tissues. Such specificity is essential for advancing our understanding of the complex interplay between ROS and its context-dependent and nuanced effects, whether beneficial or detrimental, which may ultimately inform the development of targeted therapeutic strategies and offer deeper insights into tissue-specific mechanisms underlying aging phenotypes and their associated hallmarks. Complex I ROS as a critical modulator of innate immune signaling and inflammation, but more work is needed to distinguish cell type specific roles from those of complex III. Further, immune activation is both critical and beneficial for survival of injury or illness, so ablation of complex I ROS in attempts to address chronic inflammation may result in other unintended consequences.

An increase in ROS production and a reduction in NAD⁺ concentrations is generally associated with aging and age-related diseases. Rimal et al. investigated brain samples from Drosophila of different ages and observed increased mitochondrial ROS levels and a decreased NAD⁺/NADH ratio in aged flies. Using CPT, a small molecule that binds to complex I subunit NDUFS3 and alters its interaction with other proteins within the soluble matrix arm of complex I which is involved in electron transfer, the study demonstrated that inhibition of RET, rescued age-related changes in ROS levels and NAD⁺/NADH ratio. Furthermore, RET inhibition maintained proteostasis, improved mitochondrial function, extended both healthspan and lifespan, and mitigated age-related loss of gut integrity [58]. Complementary evidence for the contribution of complex I-derived ROS to cellular pathology was shown by using S1QELs (S1QEL1 and S1QEL2), cell-permeant compounds that selectively suppress superoxide and H₂O₂ production at the ubiquinone-binding site of complex I. S1QEL-mediated inhibition of complex I ROS generation mitigated ROS-induced intestinal stem cell hyperplasia in Drosophila in vivo [126]. Additionally, S1QELs improved post-ischemic recovery of cardiac function in perfused mouse heart, consistent with the known implication of excessive RET-ROS in ischemia-reperfusion injury [76]. Collectively, these findings highlight the role of complex I-derived ROS to both age-related tissue dysfunction and acute cellular stress signaling [76,126].

Complex I-derived ROS has been implicated in nutrient-sensing pathways, particularly through redox-dependent modulation of AMP-activated protein kinase (AMPK), a key regulator of cellular energy homoeostasis that aligns mitochondrial ATP generation with cellular energy demands [127]. Hinchy et al. demonstrated, using MitoPQ, to generate ROS at complex I in HEK 293T cells, that complex I-derived ROS activates AMPK indirectly primarily, though not entirely, through ROS-induced alterations in the ATP/ADP ratio rather than through direct redox modifications of AMPK itself [60]. This mechanistic distinction positions complex I-derived ROS as an upstream bioenergetic signal that indirectly influences AMPK, rather than serving as a direct activator. With age, AMPK activity declines [128,129], while dysregulated ROS production at complex I disrupts the ATP/ADP ratio. This contributes to impaired AMPK signaling, leading to failures in mitochondrial biogenesis, mitophagy, and metabolic homoeostasis [130]. Thus, complex I-derived ROS serve as a mechanistic link between mitochondrial dysfunction and the failure of nutrient-sensing fidelity in aging cells [2] (see Fig. 5).

Fig. 5.

Complex I-derived ROS and health benefit

Schematic summary of discussed experimental findings linking complex I-derived ROS to health outcomes across model organisms and mammalian systems. The graph illustrates the non-linear relationship between complex I ROS and its biological impact, plotted as health benefit from low to high (y-axis) against biological response from adaptive over context-dependent, age-decline to pathological (x-axis). The adaptive zone (green) encompasses conditions in which complex I activity/ROS supports beneficial signaling outcomes. The context-dependent/age-decline zone (yellow) reflects conditions in which complex I function/ROS is altered by aging or disease context, producing intermediate or declining health outcomes compared to the maximal health benefit. The pathological zone (red) encompasses conditions of complex I dysfunction/excessive ROS production that drive aging phenotypes and detrimental health outcomes. Numbered circles indicate individual experimental findings detailed next to the graph with a description of each finding, organized by outcome zone. Figure was generated using Biorender.com.

5. Complex II

5.1. ROS production by complex II

Complex II, also known as succinate:ubiquinone oxidoreductase (succinate dehydrogenase, SDH), holds a unique role in cellular metabolism as the only enzyme involved in both the tricarboxylic acid (TCA) cycle and the ETC [131]. Unlike complexes I, III, and IV, complex II is — with rare exceptions — entirely encoded by the nuclear genome and does not contribute to proton translocation across the inner mitochondrial membrane [131]. However, its catalytic function is essential for aerobic energy metabolism and mitochondrial redox homeostasis [131,132].

Structurally, human complex II is a heterotetrameric protein embedded in the inner mitochondrial membrane, composed of four subunits - SDHA, SDHB, SDHC, and SDHD - along with cofactors that are critical for its catalytic and electron-transfer functions (Fig. 6A and B). The SDHA subunit contains a covalently bound flavin adenine dinucleotide (FAD) cofactor at its active site, enabling succinate oxidation and serving as the primary site of electron entry into the complex. The SDHB subunit hosts three iron-sulfur clusters that form a redox relay, transferring electrons from FAD to ubiquinone. The SDHC and SDHD subunits form the hydrophobic transmembrane anchor, embedding the complex in the inner mitochondrial membrane and coordinating a single heme b prosthetic group, which is thought to act as a redox buffer to minimize electron leakage and ROS generation [[131], [132], [133]] (Fig. 6 A).

Fig. 6.

Structure and ROS generation sites of complex II

A) Ribbon representation of the human complex II crystal structure illustrating the four subunits and key factors. SDHA (green) contains the flavin adenine dinucleotide (FAD, II_F, yellow), which serves as the primary electron entry point through succinate oxidation and the principal site of ROS generation. SDHB (blue) houses three iron-sulfur (Fe–S) clusters (magenta) that form the electron relay between the FAD cofactor and the ubiquinone-binding site (II_Q, neon green). SDHC (orange) and SDHD (purple) form the transmembrane anchor, coordinating the heme b prosthetic group (cyan) and the II_Q site, which represents a secondary site of ROS generation of complex II. B) Schematic of complex II electron flow and ROS generation. Succinate is oxidized to fumarate at the SDHA subunit, releasing electrons that pass through the Fe–S cluster relay to reduce ubiquinone (CoQ) at the II_Q site. Figure was generated using Biorender.com and ChimeraX, PDB structure 8GS8.

Within the TCA cycle, complex II catalyzes the oxidation of succinate to fumarate at the SDHA active site, releasing two electrons that reduce the covalently bound FAD to FADH₂. These electrons are then transferred through the chain of iron-sulfur clusters, to reduce ubiquinone to ubiquinol, which then delivers electrons to complex III during the forward electron transfer [43,132].

The role of complex II in ROS production was historically underappreciated compared to the well-established contributions of complexes I and III [43]. However, it is now recognized that complex II generates both superoxide and H₂O₂, with proportions influenced by the oxidation state of the iron-sulfur cluster center and occurs exclusively in the matrix [31,134]. ROS generation at complex II occurs at two sites: the flavin site of complex II (II_F) and the ubiquinone-binding site (II_Q), with their relative contributions determined by substrate availability, the redox state pf the CoQ pool, and the degree of electron flow through the complex [43,135] (Fig. 6 B). The flavin site of complex II requires two conditions for substantial ROS production: the reduction of the flavin by an electron donor and an unoccupied site to allow oxygen access. Under conditions of high succinate availability, the flavin site is occupied by succinate, minimizing oxygen access and ROS production despite sufficient electron supply [31,134,136]. As succinate levels decline, the flavin site becomes less occupied while electron supply remains adequate, leading to peak ROS production [31,43,137]. This highlights the intricate and context-dependent nature of ROS production, particularly here in the case of complex II-derived ROS. Succinate plays a dual role by both supplying electrons to reduce the flavin and occupying the active site, creating a tension between these requirements and resulting in a bell-shaped relationship between succinate concentration and ROS generation [31,43,137]. Site-specific complex II inhibitors have provided important mechanistic insight into the relative contributions of the IIF-site and the ubiquinone-binding site [43,137]. Inhibitors targeting the succinate-binding site, such as malonate, reduce ROS production by blocking FAD reduction and preventing electron entry into the complex. In contrast, inhibitors targeting the ubiquinone-site, such as TTFA, enhance ROS generation by reducing FAD and blocking forward electron transfer to CoQ, thereby increasing the probability of electron leak at both sites [43,138].

Importantly, under metabolic stress conditions like ischemia or hypoxia, tissue succinate concentrations often fall within the range of maximal ROS output from site II_F, highlighting its potential as a significant but context-dependent source of ROS production. ROS production from complex II has been associated with aging and age-related disorders, contributing to oxidative stress and mitochondrial dysfunction [31,139,140].

5.1.1. Complex II-derived ROS and its impact on aging and aging hallmarks

Complex II is now recognized as a physiologically and pathologically significant source of ROS, directly influencing aging phenotypes and the hallmarks of aging. The mev-1 gene in C. elegans, encoding the cytochrome b large subunit (SDHC) of complex II, has been a foundational genetic model for investigating these connections [141]. The mev-1(kn1) mutant, which carries a loss-of-function defect in SDHC, exhibits shortened lifespan, hypersensitivity to oxidative stress, and rapid accumulation of aging markers [[140], [141], [142], [143], [144]]. Senoo-Matsuda et al. demonstrated that mev-1 mutants overproduce ROS under normoxic conditions, with levels further elevated under hyperoxia compared to wildtype animals, explaining the oxygen-dependent acceleration of aging previously observed in these mutants [143]. Importantly, the study confirmed that the increased ROS production originates from complex II itself rather than from complexes I and III, directly implicating the ubiquinone-binding site of complex II as the ROS generating site in this model [143]. Studies in yeast have further validated that the ubiquinone-binding site of complex II can act as a source of superoxide [135]. Additionally, mev-1 mutants show reduced glutathione levels under normoxic conditions, highlighting the dual impact of complex II dysfunction, increased ROS generation and weakened antioxidant defenses, suggesting that complex II-derived ROS depletes the glutathione pool, and compromising the cell's capacity to buffer oxidative stress [143]. These mutants also exhibit increased lactate levels, indicative of lactic acidosis, a hallmark of mitochondrial diseases caused by impaired oxidative phosphorylation and increased reliance on glycolysis for ATP production [143]. The mev-1 mutant additionally displays mitochondrial structual abnormalities, loss of membrane potential, altered CED-9 and Cyt-1 levels under hyperoxia, and contain ced-3 and ced-4-dependent apoptotic cells. These defects collectively explain their failure of mev-1 mutants to complete embryonic development under hyperoxia and their reduced lifespan [145]. Furthermore, complex II-derived ROS has been linked to the induction of nuclear DNA damage and subsequent mutagenesis of a chromosomal gene [146], establishing a direct mechanistic connection between complex II-derived ROS and genomic instability, an established hallmark of aging [2]. Together, these findings link complex II dysfunction and ROS production to aging, DNA damage, and mitochondrial disease pathophysiology [143,145,146].

The relevance of complex II-derived ROS to aging extends beyond the SDHC subunit to other complex II components. Huang and Lemire investigated the molecular and catalytic effects of mutations in the sdhb-1 gene, encoding the iron-sulfur subunit of complex II [147]. They targeted a specific residue (Pro 211) located near the ubiquinone-binding site, which is conserved in humans (Pro 197), where it is associated with tumorigenesis [147], likely through SDH-dysfunction induced ROS production and HIF-1α [148]. The sdhb-1 mutants display hypersensitivity to oxidative stress, reduced lifespan, impaired respiration, and increased ROS levels, phenocopying the mev-1 mutants and demonstrating that disruption of structurally distinct subunits of complex II converges on common aging phenotypes [141,147]. The authors confirmed that ROS originated specifically from the ubiquinone-binding site of complex II [147], consistent with the findings of the mev-1 studies, reinforcing the ubiquinone-binding site of complex II as ROS production site [[140], [141], [142], [143], [144]]. The C184Y SDHB mutation in yeast (corresponding to C91Y in human SDHB) suppresses complex II activity, likely by disrupting one of the iron-sulfur clusters [131], leading to increased ROS production and mitochondrial DNA mutability [149], further linking complex II-derived ROS to genomic instability. The conservation of these findings extends to Drosophila. Walker et al. reported that Drosophila mutants carrying a defect in the sdhB gene, encoding the same iron-sulfur subunit affected in C. elegans sdhb-1 mutants, are hypersensitive to oxygen, paralleling the oxygen-sensitive phenotypes of both C. elegans mev-1 (SDHC) and sdhb-1 (SDHB) mutants and demonstrating cross-species conservation of the complex II ROS-oxygen sensitivity axis [142,147,150]. Pathological analysis of flight muscle revealed mitochondrial structural abnormalities in sdhB mutants. Biochemical studies confirmed that the defect was specific to complex II, with all other respiratory complexes remaining intact, identifying complex II dysfunction as the main contributor to the observed phenotypes. These sdhB mutants exhibit increased mitochondrial hydrogen peroxide production, and shortened lifespan with age-related behavioral decline [150]. A further transgenic fly strain overexpressing SdhC^I71E, a mutation corresponding to the mev-1 mutation in C. elegans, showed a reduction in lifespan and accumulated protein carbonyl [151], indicating that elevated complex II-derived ROS drives oxidative protein modification and contributes to accelerated aging [150,151].

Besides invertebrate model organisms, the significance of complex II-derived ROS extends to mammalian systems, where SDHC and SDHD mutations have been directly linked to genomic instability, premature senescence, and oncogenic transformation. A study by Slane et al. demonstrated that a single-base mutation in SDHC in Chinese hamster lung fibroblasts (B9 cells) caused aneuploidy and genomic instability, which were associated with elevated superoxide levels. Furthermore, B9 cells showed increases in the glutathione/glutathione disulfide ratio, glucose consumption, and sensitivity to glucose deprivation-induced cytotoxicity, the latter has been linked to metabolic stress [55,152]. The expression of wildtype hSDHC in B9 cells restored the wildtype phenotype by reversing prooxidant production, glucose consumption, sensitivity to glucose deprivation-induced cytotoxicity, and aneuploidy, demonstrating that complex II dysfunction can be both necessary and sufficient for the observed phenotypes. The authors suggested that these effects were consistent with oxidative stress driven by increased ROS production at complex II [55]. Additionally, a study by Owens et al. employed site-directed mutagenesis to introduce nonsense point mutation in human SDHD, disrupting the CoQ-binding site of complex II [153]. In Chinese hamster fibroblasts (B1 cells), this mutation led to elevated ‘steady-state-levels’ of superoxide, which significantly contributed to increased mutation rates and frequency driven by both superoxide and its dismutation product hydrogen peroxide. These findings support the hypothesis that SDHD mutations may facilitate carcinogenesis by promoting genomic instability through complex II-derived ROS levels [153]. Comparable effects were observed in yeast models, where SDHD mutations were linked to increased ROS production, genomic instability, and reduced chronological lifespan [154], further demonstrating the evolutionary conservation of complex II-derived ROS as a driver of genomic instability and aging across model systems.

Complex II dysfunction has been identified as a significant source of ROS, contributing to oxidative damage and premature cellular senescence in hematopoietic stem cells (HSCs) [155]. Using a mouse model with a targeted missense mutation in SDHC (Sdhc^V69E), Harada et al. demonstrated that disruption of complex II function leads to elevated ROS levels and increased DNA damage during oxidative phosphorylation. These defects were associated with aging-related changes in the hematopoietic system, including reduced white blood cell counts with myeloid-skewing of differentiation, macrocytic anemia, and thrombocytosis. Aged mutant HSCs exhibited lower mitochondrial membrane potential, indicating partial uncoupling due to ROS-induced membrane damage or due to ETC impairment, and under replicative stress from transplantation showed premature hematopoietic senescence and impaired long-term reconstitution capacity. Together, these findings demonstrate that complex II dysfunction drives excessive ROS production and DNA damage, causing premature cellular senescence in HSCs, ultimately compromising hematopoietic function and aging [155]. A further study by Ishii et al. used a transgenic mouse fibroblast NIH3T3 cell line expressing SDHC E69 to link complex II dysfunction and complex II-derived ROS to carcinogenesis and tumorigenesis [156]. This cell line has increased cytoplasmic protein carbonyl content, which is a marker for irreversible oxidative protein modifications and oxidative stress, and elevated 8-OH-deoxyguanine, a DNA marker of oxidative stress. Furthermore, the cells showed an increased mutation frequency compared to wildtype cells and elevated superoxide levels, confirming that SDHC E69-driven complex II dysfunction generates ROS, which damaged both DNA and proteins, driving mutagenesis and oncogenic transformation, which links mitochondrial dysfunction from complex II-derived ROS to apoptosis and tumorigenesis [156].

Direct evidence for the importance of complex II dysfunction to human aging comes from primary human cell studies, extending the model organism and cell line findings into a physiologically relevant human context. In human studies, Bowman and Birch-Machin investigated age-related changes in complex II activity using primary human skin cells [157]. Their findings revealed a significant age-dependent decline in complex II activity in skin fibroblasts from donors aged 6-72 years, which was absent in skin keratinocytes, suggesting that the age-dependent decline in complex II activity is cell-type specific rather than a general aspect of human skin aging. Reduced transcript expression and protein levels of the catalytic subunits SDHA and SDHB were detected with age in skin fibroblasts, which is consistent with other studies, that showed that increased ROS levels lead to decreased SDHB protein expression and in complex III activity in mouse heart [158] and skin [159]. Notably, complex II activity decline was specifically associated with senescent skin cells, suggesting that complex II may play a role in both cellular senescence and the overall aging phenotype [157]. The authors speculate that the age-dependent decline in complex II activity may result from increased ROS levels, due to reduced cellular defenses with age, which oxidatively damage nuclear DNA-encoded subunits and complex II-associated proteins, leading to further complex II dysfunction and amplified ROS production. Reduction in complex II activity and amplified ROS production may directly contribute to cellular aging by increasing electron leak, damaging cellular components, and impairing tissue function [157].

Further insights into complex II-derived ROS signaling were provided by Trewin et al., who demonstrated that superoxide generation from complex II is spatially regulated and elicits distinct signaling outcomes depending on the sub-organellar site of production [35]. Using an optogenetic approach in C. elegans, the study showed that matrix-directed superoxide production activates stress-responsive pathways, including p38 MAPK (PMK-1) signaling and SKN-1/Nrf2-dependent antioxidant transcription, collectively enhancing mitochondrial stress resistance and redox homeostasis. In contrast, superoxide generated toward the intermembrane space caused comparatively modest transcriptional responses, suggesting that the spatial localization of complex II-derived ROS, rather than its general production, influences its impact on aging-related processes. Importantly, these findings challenge the simplistic ROS dose-response model of aging, highlighting instead that the sub-organellar source of superoxide plays a critical role in determining whether ROS act as adaptive signaling molecule or contribute to oxidative damage [35]. Matrix-localized complex II–derived ROS further conferred protection against anoxia-reoxygenation stress, promoting mitochondrial stress resistance, likely through activation of PMK-1-dependent stress response genes that collectively mitigate the oxidative burst connected to reoxygenation. These findings suggest that controlled, spatially restricted ROS signaling from complex II activates conserved redox-responsive pathways that support healthy aging, by maintaining mitochondrial and cellular homeostasis under conditions of oxidative stress and may delay the onset of aging hallmarks [35].

Complex II has emerged as a critical regulator of inflammatory signaling beyond its canonical role in electron transport and energy metabolism. In inflammatory macrophages, SDH activity is required for proper activation of the STAT3–IL-10 anti-inflammatory pathway, indicating a mechanistic link between mitochondrial metabolism, redox homeostasis, and immune regulation [63]. Genetic disruption of complex II subunits impairs STAT3 tyrosine phosphorylation and markedly reduces IL-10 production following inflammatory stimulation, indicating that intact complex II function is necessary for anti-inflammatory transcriptional responses through STAT-3-dependent cytokine signaling [63]. Mechanistically, loss of SDH leads to dysregulated mitochondrial ROS accumulation, which interferes with STAT3 activation, highlighting a redox-dependent control point in cytokine signaling. These findings highlight complex II as a central immunometabolic regulator that coordinates both pro- and anti-inflammatory pathways via mitochondrial ROS signaling. Physiological complex II-derived ROS supports STAT3-mediated anti-inflammatory responses, whereas dysregulated ROS production disrupts this balance, leading to chronic inflammation. This underscores the importance of the subcellular localization, magnitude, and signaling context of complex II-derived ROS, rather than their sheer abundance, in shaping macrophage inflammatory responses and connecting mitochondrial redox dysregulation to inflammaging [63].

Collectively, these studies demonstrate that complex II dysfunction contributes to aging through diverse but converging mechanisms, primarily by driving excessive or mislocalized ROS production, which leads to stem cell exhaustion, cellular senescence, and impaired immune and stress-response signaling. The interplay of mitochondrial dysfunction, genomic instability, chronic inflammation, and disrupted stress-response signaling highlights complex II-derived ROS as a central mediator connecting and influencing multiple hallmarks of aging. Critically, the evidence reviewed demonstrates that the role of complex II-derived ROS in aging is not solely determined by its quantity, but rather by its spatial source, signaling context, and downstream targets. These factors ultimately dictate whether ROS acts as an adaptive signal beneficial for longevity or as a driver of pathological aging, further emphasizing its nuanced and multifaced role (Fig. 7).

Fig. 7.

Complex II-derived ROS and health benefits outcomes.

Schematic summary of discussed experimental findings linking complex II-derived ROS to health outcomes across model organisms and mammalian systems. The graph illustrates the non-linear relationship between complex II ROS and its biological impact, plotted as health benefit from low to high (y-axis) against biological response from adaptive over context-dependent, age-decline to pathological (x-axis). The adaptive zone (green) encompasses conditions in which complex II activity/ROS supports beneficial signaling outcomes. The context-dependent/age-decline zone (yellow) reflects conditions in which complex II function/ROS is altered by aging or disease context, producing intermediate or declining health outcomes compared to the maximal health benefit. The pathological zone (red) encompasses conditions of complex II dysfunction/excessive ROS production that drive aging phenotypes and detrimental health outcomes. Numbered circles indicate individual experimental findings detailed next to the graph with a description of each finding, organized by outcome zone. Figure was generated using Biorender.com.

6. Complex III

6.1. ROS production by complex III

Complex III, also known as cytochrome bc₁ complex, transfers electrons from reduced ubiquinone to cytochrome c through a mechanism called the Q cycle, simultaneously translocating protons across the inner mitochondrial membrane, to contribute to the mitochondrial membrane potential. Complex III is a dimer, with each monomer comprising eleven subunits encoded by both the mitochondrial and nuclear genome [160] (Fig. 8). During the Q cycle, ubiquinol is oxidized at the outer quinone-binding site (Q_o) site of complex III, releasing two electrons. One electron is transferred to cytochrome c via the Rieske iron-sulfur protein (RISP) and cytochrome c₁, while the other electron is transferred through cytochrome b to reduce ubiquinone at the inner quinone-binding site (Q_i) of the complex, regenerating ubiquinol for the next catalytic cycle [41]. For every two electrons transferred to cytochrome c, four protons are translocated across the inner mitochondrial membrane, contributing to the mitochondrial membrane potential and used by complex V to synthesize ATP (Fig. 8 B).

Fig. 8.

Structure and ROS generation sites of complex III

A) Ribbon representation of the human complex III homodimer crystal structure illustrating the key subunits and cofactors. Cytochrome b (yellow) is the central catalytic subunit, the outer ubiquinone binding site (Q_o, magenta) and the inner ubiquinone binding site (Q_i, cyan), the Rieske iron-sulfur protein (RISP, orange) and cytochrome c₁ (blue). B) Schematic of electron flow, proton translocation, and ROS generation of complex III. Ubiquinol is oxidized at the Q_o site, releasing two electrons. One electron is transferred to cytochrome c (purple) via RISP and cytochrome c₁, while the other reduces ubiquinone at the Q_i site. ROS can be generated at both the Q_o site and the Q_i site. Figure was generated using Biorender.com and ChimeraX, PDB structure 5XTE.

The primary site of complex III-derived ROS production is the Q_o site, where a transient semiquinone radical forms, which can react with molecular oxygen to produce ROS in the form of superoxide. Slower electron transfer increases the accumulation of semiquinone intermediates at the Q_o site, raising the probability of ROS generation [161,162]. Biomedical studies using site-specific inhibitors have identified the Q_o site semiquinone as the primary electron donor to O₂ for superoxide production within complex III [163]. Antimycin A, which blocks ubiquinone reduction at the Q_i site, enhances ROS production, consistent with accumulation of a semiquinone intermediate at the Q_o site, though the precise mechanisms of semiquinone stabilization remain debated [[164], [165], [166], [167]]. In contrast stigmatellin, which binds to the Q_o site and prevents ubiquinol docking, effectively eliminates complex III-derived superoxide [168]. Myxothiazol, which binds to the proximal niche of the Q_o site, does not fully suppress superoxide production and can even stimulate it by permitting semiquinone formation at the proximal Q_o domain, while preventing its oxidation and release [168].

Unlike other sites in the ETC, complex III can release superoxide into both the mitochondrial matrix and the intermembrane space due to the location of the Q_o site at the outer face of the inner mitochondrial membrane [27]. ROS production at complex III is increased under conditions that slow electron flow through the Q cycle, including high mitochondrial membrane potential, low ATP demand, or pharmacological and pathological inhibition of electron transfer, all increase semiquinone residence time at the Q_o site [31,41,169].

ROS generated by complex III serve as critical signaling molecules in various physiological processes, with one of the most extensively characterized roles being the regulation of the cellular hypoxic response. Under hypoxic conditions, complex III-derived ROS play a key role in activating hypoxia-inducible factors (HIFs), which regulate genes essential for cellular metabolism and survival [170,171]. Klimova & Chandel 2008 demonstrated that targeting complex III-derived ROS modulates HIF-1α activation, establishing a direct link between ROS production and hypoxic transcriptional reprogramming signaling [172]. Beyond cellular hypoxia adaptation, complex III-derived ROS influence aging-related pathways, including oxidative stress, cellular senescence, and oncogenic signaling — for example, in melanoma cells, complex III ROS-driven HIF-1α stabilization promotes invasive growth, while inhibiting mitochondrial ROS reduced aggressive tumor behaviors, highlighting how the same signaling mechanism can drive both physiological adaptation and pathological progression, depending on the context [[173], [174], [175], [176], [177]].

While physiological complex III-derived ROS serve as spatially and temporally regulated signaling molecules, excessive or dysregulated production contributes to oxidative damage to lipids, proteins, and DNA, and influences the aging phenotype and several hallmarks of aging, which will be discussed in the following section, and highlights the context-dependent nature of site-specific mitochondrial ROS.

6.1.1. Complex III-derived ROS and its impact on aging and aging hallmarks

Aging leads to cardiac impairment due to the loss of cardiomyocytes, compromised physiological function, and increased chronic oxidative damage, particularly in mitochondria. This oxidative damage contributes to age-related cardiac diseases including atherosclerosis and congestive heart failure. Lesnefsky et al. established a mechanistic connection between aging and mitochondrial dysfunction, identifying complex III as a critical site of functional impairment in aged rat hearts. By isolating interfibrillar mitochondria (IFM) and subsarcolemmal mitochondria (SSM) from adult and aged rats, the study demonstrated that aging selectively impairs oxidative phosphorylation in IFM but not SSM, establishing a mitochondrial subpopulation-specific sensitivity to age-related dysfunction that may reflect the different energy demands of IFM and SSM [178]. This functional defect was not attributed to the loss of complex III but was instead linked to a site-specific functional alteration at complex III that becomes evident under physiological electron transport conditions [62]. In the context of aging biology, this dysfunction aligns with the hallmark of mitochondrial dysfunction and increased oxidative stress, suggesting that impaired complex III activity in IFM contributes to the aging cardiac phenotype and heightened susceptibility to ischemia–reperfusion injury [62]. Moghaddas et al. extended these findings using Fischer 344 rats (6-month-old versus 24-month-old) to investigate aging-related defects in cardiac mitochondria with greater mechanistic resolution [179]. IFM and SSM were isolated, and complex III was analyzed using Qo site inhibitors – myxothiazol, which binds to the proximal domain of the Qo site, and stigmatellin, which binds to the distal domain – to spatially localize the functional defect within the complex. H₂O₂ production was measured as a proxy complex III-derived ROS generation. In aged IFM, cytochrome b reduction increased by 40% with both inhibitors, and electron leak persisted with myxothiazol alone but was abolished with stigmatellin, localizing the ROS-generating defect to the proximal myxothiazol-binding domain of the Qo site. The structural defect within complex III resulted in a persistent electron leak during both forward and reverse electron flow, contributing to elevated oxidative stress. This suggests that the Qo site defect is a permanent structural alteration rather than a redox state-dependent condition. H₂O₂ production was significantly elevated with myxothiazol and antimycin A but not with stigmatellin, further confirming that the proximal Qo site is the source of the age-related ROS generation in cardiac IFM. In contrast, SSM showed no age-related changes, highlighting the specificity of the defect to IFM, this may be attributed to the lower energy demand of SSM, potentially resulting in decreased exposure to mitochondrial stress [[178], [179], [180]]. Together, these studies identify the proximal Qo site of complex III in cardiac IFM as the primary molecular locus of age-related mitochondrial dysfunction, linking structural alterations at this site to electron leak, increased ROS production, and the oxidative aging phenotype of cardiac tissue [62,179].

The recognition of the Qo site as source of age-related complex III dysfunction is further supported by evidence linking complex III disruption to cellular senescence, an established hallmark of aging [2,61]. Oncogene-induced senescence (OIS) is a tumor-suppressive mechanism in which oncogenic ras expression in normal human cells triggers aberrant proliferative signaling, followed by irreversible cell cycle arrest. Moiseeva et al. investigated the role of mitochondrial dysfunction in this process using normal human fibroblasts and epithelial cells. Cells expressing oncogenic ras showed increased mitochondrial mass, mitochondrial DNA content, and mitochondrial ROS production prior to the onset of senescence, accompanied by oxidative DNA damage, reduced ATP levels, and AMPK activation [61]. Critically, disruption of complex III, via knockdown of RISP, a core subunit of the Qo site where age-associated ROS production increases [41,61,179], or via pharmacological inhibition of the ETC, was sufficient to induce senescence in normal human cells in the absent of oncogene signaling [61]. This demonstrated that complex III dysfunction alone is sufficient to activate the senescence program independently of oncogenic signaling, suggesting ROS production at the Qo site as a potential contributor to the initiation of cellular senescence. The age-related Qo site defect described by Moghaddas et al. and the RISP-dependent senescence induction reported by Moiseeva et al. suggest that structural decline of the Qo site may contribute to reduced regenerative capacity with age [61,179]. However, while mitochondrial dysfunction was associated with oxidative damage and genomic instability, Moiseeva et al. did not specifically isolate complex III-derived ROS as the mediating signal, therefore other effects of RISP loss, such as altered metabolite pools or impaired electron flow, may also contribute to senescence induction. Further research is needed to clarify the specific role of complex III-derived ROS in this process [61].

In contrast to the pathological consequence of complex III dysfunction in cardiac aging and oncogene-induced senescence, research in model organisms suggests that a partial disruption of complex III function can paradoxically promote lifespan extension by triggering ROS-dependent activation of conserved stress-response pathways. A study by Feng et al. identified a mutation in the iron-sulfur protein (isp-1) of mitochondrial complex III in C. elegans, resulting in reduced oxygen consumption, decreased sensitivity to ROS, and extended lifespan [96]. Yang and Hekimi (2010) challenged the mitochondrial oxidative stress theory of aging by demonstrating that elevated mitochondrial superoxide generation, rather than reduced ROS levels, promotes longevity in isp-1 mutants [15]. Using flow cytometry of isolated mitochondria, they confirmed that superoxide levels were elevated while overall ROS levels and markers of oxidative stress remained low, a separation suggesting that superoxide functions as a spatially controlled signaling molecule rather than as a generalized oxidative stressor. Superoxide was shown to be both necessary and sufficient for lifespan extension, as antioxidant treatment with NAC and vitamin C abolished the longevity effect, while mild prooxidant treatment with paraquat mimicked it. The authors propose that during normal aging, the progressive accumulation of molecular damage gradually increases superoxide generation as a compensatory protective response, reframing the correlation between ROS and aging as a consequence of aging rather than a cause [15]. Dues et al. extended these findings by demonstrating that in isp-1 mutants, elevated ROS activate stress-response pathways, accompanied by increased expression of antioxidant enzymes [181]. Critically, deletion of superoxide dismutase genes sod-3 or sod-5 enhances oxidative stress resistance but paradoxically reduced lifespan, experimentally separating oxidative stress resistance from longevity and demonstrating that these are separable phenotypes [181]. These findings demonstrate that the extended lifespan in isp-1 mutants is not solely attributable to increased antioxidant capacity or reduced ROS sensitivity, instead emphasizing superoxide as a signaling molecule with targets that influence longevity [15,181].

Senchuk et al. investigated three long-lived mitochondrial mutants isp-1 (complex III), nuo-6 (complex I), and clk-1 (disrupted ubiquinone biosynthesis), which disrupt mitochondrial function at distinct points but share elevated mitochondrial ROS as a common feature [18]. The study demonstrated that mitochondrial ROS activates the DAF-16/FOXO transcription factor, driving its nuclear translocation and the transcription of longevity-promoting target genes. Transcriptomic analysis revealed upregulation of DAF-16 target genes in all three mutants, with patterns similar to those of daf-2 mutants, which are long-lived due to reduced insulin/IGF-1 signaling. Antioxidant treatment reduced DAF-16 nuclear translocation and target gene expression, confirming ROS as the causal signal. Genetic loss-of-function experiments demonstrated that DAF-16 is essential for the extended lifespan of all three mutants, establishing mitochondrial ROS as a key upstream driver of DAF-16-mediated longevity across mechanistically distinct models of mitochondrial dysfunction [18]. Mutations in the clk-1 gene in C. elegans slow developmental and physiological processes broadly, including the rate of aging. In yeast, the CLK-1 ortholog is essential for ubiquinone biosynthesis and respiration, and CLK-1 performs an equivalent function in C. elegans, producing ubiquinone that is essential for electron transfer at both complexes I and III [94,182]. Overexpression of CLK-1 in wildtype worms increased mitochondrial activity, accelerated behavioral rates during aging, and shortened lifespan, demonstrating that clk-1 regulates the rate of aging and providing evidence linking mitochondrial electron transport efficiency to longevity [94]. Although clk-1 does not directly encode a subunit of complexes I or III, its disruption decreases ubiquinone availability, impairing electron transport at both complexes I and III and potentially increasing electron leak and ROS production, thereby indirectly impacting the same aging pathways activated by direct mutations in complexes I and III.

Dillin et al. investigated the role of complex III activity on aging by using RNAi to knockdown cyc-1, which encodes a subunit of complex III, in C. elegans [87]. Worms subjected to cyc-1 RNAi showed reduced ATP levels regardless of whether knockdown was initiated at hatching or in adulthood. However, lifespan extension was observed only when RNAi was initiated from hatching, adult-initiated knockdown showed no longevity effect, demonstrating that the developmental timing of complex III disruption, rather than lower ATP levels in general, is the critical determinant of this longevity outcome [87]. In a large-scale RNAi screen of C. elegans genes, inactivation of T02H6.11, encoding a subunit of complex III and the C. elegans ortholog of the Drosophila gene CG17856, was identified as sufficient to extend lifespan [88]. Copeland et al. demonstrated that RNAi targeting CG17856 in Drosophila extends lifespan when initiated during development, with a threshold effect, where moderate knockdown resulted in lifespan extension, while stronger inhibition caused developmental lethality [16]. Adult-specific RNAi of CG17856 showed no significant impact on lifespan in either sex, again demonstrating the developmental timing dependence of complex III-mediated lifespan extension and the existence of a dosage threshold below which complex III disruption becomes detrimental rather than beneficial [16]. This is consistent with a mitohormetic model [17], in which moderate complex III dysfunction activates adaptive ROS signaling while severe dysfunction causes irreversible damage. Collectively, these findings demonstrate that the relationship between complex III function, its ROS production, and longevity is highly context-dependent, shaped by location, amount and timing of dysfunction, and the downstream signaling pathways engaged. Complex III-derived ROS functions as an upstream activator of conserved longevity transcription factors, but only when generated within a specific magnitude range and timed-context. Greater mechanistic specificity in defining the identity, site, magnitude and timing of complex III-derived ROS production will be essential for resolving the apparent contradictions between lifespan-extending and lifespan-shortening effects of complex III disruption across model systems.

In addition to its role in longevity regulation in model organisms, complex III-derived ROS has been directly linked to neuropathological processes and age-related tissue dysfunction in mammalian systems, highlighting the broader significance of site-specific complex III ROS in the context of human disease. Dysfunctional mitochondria represent a common and early hallmark of neurodegenerative disorders, including AD, with mitochondrial ROS serving as a key contributor to cellular dysfunction and neuropathology [66,183,184]. Using a brain-specific conditional knockout of the RISP in mouse models, Diaz et al. demonstrated that specific ETC defects lead to distinct neuronal neuropathological outcomes. Conditional RISP knockout produced elevated oxidative stress early in life and dramatically shortened lifespan, establishing that elevated complex III-derived ROS levels are sufficient to cause neuropathology even in the absence of a broader neurodegenerative disease context [185]. Barnett et al. utilized a complementary approach, using site-selective suppressors (S3QELs), astrocyte-specific AOX expression, and mitochondrial Na⁺/Ca²⁺ exchanger (NCLX) genetic knockout to demonstrate that astrocytic complex III-derived ROS are induced downstream of NF-κB activation in an NCLX-dependent manner, promoting context-specific STAT3 signaling and transcriptional reprogramming [66]. While Diaz et al. modeled the consequences of complete neuronal complex III loss [185], Barnett et al. demonstrated that selective suppression of complex III-derived ROS at the Qo site, without disrupting electron transport or ATP generation, is sufficient to mitigate dementia-associated neuropathology by alleviating maladaptive STAT3-driven inflammatory cascades [66]. Together, these findings establish complex III-derived ROS as a non-cell-autonomous immunometabolic signal in the brain, originating from astrocytes to modulate neuronal and glial inflammatory responses through STAT3-dependent transcriptional pathways. Chronic S3QEL2 treatment extended lifespan by approximately 17–20% in tauopathy mice while attenuating neuropathology, providing strong evidence that complex III-derived ROS at the Qo site represents both a mechanistic driver of brain aging and a viable therapeutic target for promoting healthy longevity [66,185].

The pathological impact of complex III-derived ROS extends beyond the nervous system, evidence also links complex III-derived ROS with diet-induced internal barrier dysfunction, and therefore, showing a connection to another important tissue axis which is involved in age-related systemic decline. Disruption of intestinal homeostasis and intestinal barrier integrity has been reported across multiple model organisms, including C. elegans, Drosophila, rodents, and humans and is linked to a systemic health decline [[186], [187], [188], [189], [190]]. Intestinal barrier dysfunction can be experimentally replicated through nutrient-rich diets, as animals fed a high-fat diet show increased oxidative stress and reduced intestinal integrity over time, with complex III-derived ROS emerging as a mechanistic contributor to this diet-induced barrier disruption, as discussed below, and is associated with decreased lifespan [65,191,192]. Watson et al. investigated the role of complex III-derived ROS in diet-induced intestinal barrier dysfunction using S3QELs, small molecules that selectively suppress superoxide generation at the Qo site of complex III without inhibiting normal electron flow, oxidative phosphorylation or other known cellular processes [65]. Feeding S3QELs to Drosophila maintained on a high-nutrient diet reduced intestinal permeability, decreased enterocyte apoptotic cell numbers, and increased median lifespan by 10-20%. Comparable protection was observed in mice fed a high-fat diet, where S3QEL treatment diminished diet-induced increases in intestinal permeability. Critically, S1QELs, which suppress complex I-derived ROS at site I_Q, did not protect against diet-induced intestinal permeability, confirming the site-specificity of complex III-derived ROS in driving intestinal barrier disruption. Together, these findings demonstrated that ROS production at complex III in enterocytes is a key driver of diet-induced intestinal barrier disruption in both flies and mice, mechanistically connecting this site-specific ROS production to the breakdown of a critical epithelial barrier for maintaining systemic homoestasis [65]. The therapeutic potential of S3QEL-mediated Qo site ROS suppression extends beyond intestinal protection. Intestinal barrier dysfunction has been mechanistically linked to multiple hallmarks of aging, including dysbiosis, chronic inflammation, and dysregulated intercellular communication, suggesting that complex III-derived ROS in enterocytes may represent an upstream initiating signal in a cascade of systemic age-related decline [188,193,194]. The connection of complex III-derived ROS with intestinal aging, neuroinflammation, and metabolic disease positions this specific production site as a broadly significant therapeutic target. S3QELs offer a mechanistically precise approach for investigating and potentially alleviating site-specific ROS contributions to age-related pathologies.

The immunomodulatory function of complex III-derived ROS is not limited to the intestinal epithelium; it also plays a critical role in regulating inflammatory cytokine production, including the anti-inflammatory cytokine Interleukin-10 (IL-10), which resolves acute inflammation and limits immune responses, and the pro-inflammatory cytokine IL-1β. Zotta et al. demonstrated that S3QEL 1.2 suppresses IL-10 production in LPS-activated macrophages by inhibiting c-Fos, a subunit of activator protein 1 (AP1) whose activity is required for IL-10 transcription downstream of TLR stimulation [[195], [196], [197]]. The canonical complex III inhibitor myxothiazol produced an equivalent effect, confirming that complex III-derived ROS at the Qo site, rather than off-target effects of S3QEL 1.2, is the physiological driver. In vivo, S3QEL 1.2 reduced IL-10 levels in mice, slowed B16F10 melanoma growth, and promoted survival, consistent with complex III-derived ROS supporting immunosuppressive tumor microenvironment through IL-10-mediated suppression of antitumor CD8⁺ T-cell responses and suggesting its potential for boosting antitumor immunity [195,198]. In a distinct but complementary context, Zhang et al. demonstrated that complex III activity supports IL-10 transcription during efferocytosis in the context of myocardial infarction through NAD⁺ regeneration and a SIRT1/PBX1 signaling cascade, independently of ROS production [199]. Extending these findings, Stoolman et al. showed that complex III-deficient macrophages exhibited increased susceptibility to influenza A infection and LPS-induced endotoxic shock, attributed to impaired IL-10 release. Critically, expression of AOX, which restores NAD⁺ regeneration and electron transport without generating superoxide, failed to rescue IL-10 secretion, demonstrating that complex III-derived ROS at the Qo site, rather than electron flow or NAD⁺ regeneration per se, is specifically required for IL-10 release following TLR stimulation [200]. Together, these finding are mechanistically significant as they demonstrate that complex III can regulate IL-10 production through two distinct and separable mechanisms, ROS-dependent c-Fos/AP1 activation and ROS-independent NAD⁺/SIRT1/PBX1 signaling, whose relative contributions may vary depending on the distinct inflammatory context and cell type [195,199,200].

Beyond IL-10 regulation, complex III-derived ROS also directly activates pro-inflammatory pathways. Zhou et al. demonstrated that treatment of macrophages with the complex I inhibitor rotenone or the complex III inhibitor antimycin A increased mitochondrial ROS and triggered dose-dependent IL-1β secretion through NLRP3 inflammasome activation [59]. Furthermore, impairment of autophagy and mitophagy led to accumulation of damaged, ROS-generating mitochondria and spontaneous inflammasome activation, establishing a direct mechanistic link between impaired mitochondrial quality control and chronic inflammasome-driven inflammation [59]. However, further research is required to distinguish the specific contributions of complex I- and complex III-derived ROS to NLRP3 activation, as their mechanistic roles may differ significantly, a distinction highlighted by the divergent downstream effects of S3QELs versus S1QELs observed across multiple studies. These findings collectively highlight the dual and context-dependent role of complex III-derived ROS in macrophage immunobiology, where ROS are simultaneously essential for protective anti-inflammatory IL-10 responses and capable of driving pathological NLRP3 inflammasome activation [59,195,199,200]. With aging, the progressive decline in mitophagy and ETC integrity [59] shifts this balance from protective immunoregulation toward chronic inflammation, as excessive complex III-derived ROS drives constitutive IL-1β secretion through NLRP3 activation [59], while the context-dependent mechanisms sustaining IL-10, through c-Fos/AP1 activation [195] and NAD⁺/SIRT1 signaling [199], and PKA-dependent secretory trafficking [200], become increasingly impaired. This mechanistic framework provides a molecular explanation for how mitochondrial aging drives inflammaging and positions complex III as both a central mediator of age-related immune dysregulation and a promising therapeutic target for promoting healthy longevity.

Building on the role of complex III-derived ROS in inflammation, its impact extends to innate and adaptive immune activation. Cross-presentation, the presentation of exogenous antigens onto major histocompatibility complex class I molecules for CD8⁺ T-cell activation, is crucial for antiviral and antitumor immunity. Oberkampf et al. identified a regulatory pathway linking complex III-derived ROS to antigen cross-presentation in TLR-activated plasmacytoid dendritic cells (pDCs) through ROS production. Reduction of mitochondrial ROS levels significantly impaired the ability of pDC capacity to activate CD8⁺ T-cell responses following immunization, while cytokine or chemokine production remained unaffected. Using S3QELs to specifically target ROS production at the Qo site of complex III, the study confirmed that complex III-derived ROS regulates the cross-presentation capacity of pDCs and the induction of CD8⁺ T-cell-mediated immune responses, mechanistically distinguishing this pathway from NOX2-dependent ROS in conventional dendritic cells and establishing a cell-type-specific role for mitochondrial ROS in antigen processing [198]. Complementing these findings, Sena et al. demonstrated that complex III-derived ROS is required for CD4⁺ T-cell activation through NFAT signaling and IL-2 induction in vitro, and for antigen-specific CD4⁺ and CD8⁺ T-cell expansion in vivo in mice [201]. Mechanistically, complex III-derived ROS may support T-cell activation by sustaining the elevated mitochondrial membrane potential and ROS production required for nuclear factor of activated T-cells (NFAT), a redox-sensitive step in T-cell receptor signaling [201]. Together, these findings provide mechanistic insight into how complex III-derived ROS regulates both innate immune cross-presentation in pDCs and adaptive T-cell activation. Furthermore, the well-documented age-related decline in mitochondrial function may progressively impair these ROS-dependent immune signaling pathways, reducing the capacity for effective antiviral and antitumor immune responses and contributing to immunosenescence, the age-related decline in immune competence, that is linked to increased infection rates, cancer and chronic inflammation [2,202].

Beyond its influence on immune activation and inflammation, complex III-derived ROS plays a crucial role in stem cell differentiation and maintenance, processes whose dysregulation directly contributes to the aging hallmark of stem cell exhaustion [2]. Tormos et al. demonstrated that primary human mesenchymal stem cells (MSCs) exhibit an early increase in mitochondrial biogenesis, metabolism, and ROS production during adipogenic differentiation, dependent on mTOR signaling [64]. Previous research has shown that stem cells undergo an increase in mitochondrial biogenesis and metabolism upon differentiation [203]. Genetic manipulation of complex III revealed that complex III-derived ROS is specifically required for activation of the PPARγ transcriptional machinery and initiation of adipocyte differentiation. This finding extends beyond the role of energy supply, as complex III-derived ROS not only supports the energetic demands of differentiation but also regulates the transcriptional program determining the cell fate [64]. In contrast, pathological or dysregulated complex III-derived ROS can impair rather than support stem cell function, contributing to stem cell. Anso et al. demonstrated that conditional inactivation of RISP in fetal mouse HSCs impaired differentiation and caused anemia and prenatal lethality. RISP-null fetal HSCs and progenitors exhibited increased DNA and histone methylation due to increased levels of 2-hydroxyglutarate, a metabolite that inhibits DNA and histone demethylases. In adult HSCs, RISP inactivation disrupted respiration, causing a loss of quiescence, severe pancytopenia, and lethality. Together, these findings established that complex III function is not required fetal or adult HSC proliferation but essential for both fetal HSC differentiation and adult HSC quiescence maintenance, position complex III dysfunction as a direct contributor to the stem cell exhaustion hallmark of aging through both epigenetic and bioenergetic mechanisms [204] (Fig. 9).

Fig. 9.

Complex III-derived ROS and health benefits outcomes.

Schematic summary of discussed experimental findings linking complex III-derived ROS to health outcomes across model organisms and mammalian systems. The graph illustrates the non-linear relationship between complex III ROS and its biological impact, plotted as health benefit from low to high (y-axis) against biological response from adaptive over context-dependent, age-decline to pathological (x-axis). The adaptive zone (green) encompasses conditions in which complex III activity/ROS supports beneficial signaling outcomes. The context-dependent/age-decline zone (yellow) reflects conditions in which complex III function/ROS is altered by aging or disease context, producing intermediate or declining health outcomes compared to the maximal health benefit. The pathological zone (red) encompasses conditions of complex III dysfunction/excessive ROS production that drive aging phenotypes and detrimental health outcomes. Numbered circles indicate individual experimental findings detailed next to the graph with a description of each finding, organized by outcome zone. Figure was generated using Biorender.com.

7. Conclusion

The evidence reviewed across complexes I, II, and III establishes that mitochondrial ROS production is not a uniform process but a spatially and mechanistically diverse phenomenon, with its impact on aging shaped by the specific ETC site of origin, the direction of electron flow, the magnitude of ROS production and the temporal dynamics of its production. These parameters collectively dictate whether mitochondrial ROS function as adaptive longevity signals, activating conserved stress-response pathways, extending lifespan, and supporting cellular homeostasis, or as driver of pathological aging, promoting aging phenotypes and hallmarks such as genomic instability, chronic inflammation, and cellular senescence. The evidence presented here highlights that it is no longer scientifically sufficient to attribute aging phenotypes simply to ‘mitochondrial ROS’ without determining critical factors such as the specific ETC site of origin, the electron flow direction, type of ROS generated, and the cellular context in which production occurs. This context-dependence fundamentally determines biological outcomes, and this precision must be consistently reflected in how mitochondrial ROS are discussed, measured, or therapeutically targeted.

Complexes I, II, and III each contribute to aging phenotypes through mechanistically distinct ROS-generation processes and engage overlapping aging hallmarks through distinct downstream signaling pathways. The same aging hallmarks can arise from mechanistically distinct upstream ROS signals, depending on which complex is disrupted and where ROS is generated. Notably, however, while certain hallmarks are more extensively documented for specific complexes in the current literature, this does not necessarily indicate a reduced biological impact at other ROS production sites, it emphasizes the need to examine site-specific ROS production in greater detail rather than focusing solely on overall involvement. Similarly, the fact that different ETC sites can influence the same aging hallmark does not diminish the importance of site-specific attribution. Instead, it highlights that the same hallmark can be driven by mechanistically distinct upstream signals, each requiring a distinct therapeutic approach.

Mitochondrial dysfunction plays a central and bidirectional role within the network of aging hallmarks, acting both as an upstream driver of ROS-dependent hallmark progression and as downstream result of cellular decline at the hallmark level, establishing a cycle in which ROS-induced mitochondrial damage amplifies ROS production and accelerates aging across multiple hallmark pathways simultaneously. While mitochondrial ROS are not classified as a hallmark of aging, as they do not fulfill the three defining criteria, they serve as essential upstream mediators and downstream amplifiers of hallmark progression across the entire aging network [2,32,38,39].

The cross-species convergence of the evidence reviewed here — spanning C. elegans, Drosophila, yeast, rodent models, and human primary cells — strengthens the translational significance of the site-specific ROS framework presented here. This suggests that the mechanistic principles identified represents conserved aspects of mitochondrial biology rather than model organism-specific phenomena. It is important to acknowledge that ROS generated at sites beyond complexes I, II, and III, including the pyruvate dehydrogenase complex, α-ketoglutarate dehydrogenase complex, and monoamine oxidases, as well as non-mitochondrial ROS including from NADPH oxidase, peroxidase, and xanthine oxidase, may similarly influences aging phenotypes and hallmarks [25,31,33]. The biological effects of ROS are critically dependent on their precise location of production, within tissues, cell types, and subcellular compartments, and the site-specific framework further highlights the need to refine the language and approach in research, enabling more precise and detailed investigations.

To address this precision gap, recent innovations in genetically ROS-sensitive biosensors, including roGFP2-based probes for mitochondrial redox state and HyPer7 for compartment-specific H₂O₂, have significantly enhanced our ability to detect and track ROS production at defined cellular sites, offering deeper insights into their dynamics and functional roles, that indirect pharmacological approaches cannot match [205]. These tools, such as optogenetics [206,207], chemogenetics [208], and photosensitizer [209], are beginning to provide researchers with the ability to control and manipulate the spatial and temporal ROS generation at specific cellular sites, offering new insights on their contributions to cellular processes and aging phenotypes. Studies using an optogenetic approach and biosensors like HyPer7 have been used to precisely generate ROS in defined mitochondrial microdomains, revealing how ROS diffusion and transient mitochondrial hyperfusion are influenced by localized ROS production. By using HyPer7, researchers further investigated mitochondrial H₂O₂ in mammalian cells. They observed that mitochondria release H₂O₂ directly at their surface and into the cytosol, forming steep gradients controlled by cytosolic peroxidoxins. Such studies highlight the importance of spatiotemporal properties of ROS in determining their biological effects and providing insights into the role of ROS in health and disease [205,210,211]. Despite these advances, much remains to be understood about the sources of ROS, their movement within cells, and their biological targets. Research has begun to map ROS diffusion dynamics and identify key protein residues susceptible to reversible oxidation, which are critical for redox signaling and cellular responses. For instance, studies have demonstrated that specific thiol residues in mitochondrial proteins can act as mediators of redox signaling, influencing processes such as hypoxic responses and cellular adaptation. These findings underscore the complexity of ROS biology, where the interplay between ROS generation, diffusion, and target modification shapes cellular outcomes [212,213].

In addition to controlling ROS production, the field is also exploring how ROS modifications impact aging phenotypes, with a focus on identifying biologically relevant targets and understanding their roles in age-related processes. While many ROS modifications occur through reversible oxidation of protein cysteine residues, not all cysteines are equally susceptible or biologically significant. Determining which targets are most relevant for aging remains a key challenge. Future research should integrate advanced tools such as biosensors, genome editing, and redox-sensitive models to achieve precise control and mapping of ROS-driven mechanisms. By using redox proteomics, Bleier et al. showed that complex I- and complex III-derived ROS modify distinct, non-overlapping sets of target proteins, revealing that generator-specific ROS signaling is encoded at the level of protein target identity [40]. Redox proteomics approaches — like the tool Oximouse, which mapped the mouse cysteine redox proteome across multiple tissues in vivo — identified tissue-specific redox networks, mechanisms of redox signaling, including electrostatic gating, and disease networks that change with aging, linking redox dysregulation to tissue aging. Oximouse provides a key resource for studying redox regulation in physiology and aging [214].

The development of site-specific pharmacological tools, including S1QELs and S3QELs, demonstrates the therapeutic potential of the precision framework. Thes compounds have shown significant biological effects in disease-relevant models. S1QELs inhibit complex I-derived ROS production and showed reduced ROS-induced intestinal stem cell hyperplasia in Drosophila [126] and improved post-ischemic cardia recovery in perfused mouse hearts [76]. S3QELs inhibit complex III-derived ROS production and preserved intestinal barrier integrity and increased lifespan in Drosophila and mice on high-fat diets [65], and extended lifespan in tauopathy mice while reducing neuropathology [66]. These results provide proof-of-concept that site-specific understanding of mitochondrial ROS translates directly into therapeutic opportunities.

This review and the evidence presented here highlights that the influence of mitochondria ROS on aging is determined not only by how much ROS is produced, but by where it is produced, the direction of the electron flow, the time when it is produced, and what the cell does with that oxidative signal. Advancing our understanding of aging and developing effective interventions for age-related diseases requires consistently applying this precision. This shift moves the field beyond the concept of ‘mitochondrial oxidative stress’ towards a site-specific, mechanistically detailed understanding of how ROS drives the aging process.

Disclosures

Declaration of competing interest: APW is listed as an inventor on a patent application and a co-founder of a company that uses the technology. The other authors declare that they have no conflicts of interest that could influence the contents of this article.

Funding

Work in the laboratory of A.P.W. is supported by grants from the National Institutes of Health (R01NS092558, R01NS115906, and R21AG085324), and the Alzheimer's Association (23AARG-NTF-1023159). A.M-E. and K·S.M. are supported by T32 ES007026.

CRediT authorship contribution statement

Annika Müller-Eigner: Conceptualization, Visualization, Writing – original draft, Writing – review & editing. Elena Morin: Writing – original draft. Katherine S. Morton: Writing – original draft. Andrew P. Wojtovich: Funding acquisition, Writing – review & editing.

Declaration of competing interest

Declaration of competing interest: APW is listed as an inventor on a patent application and is a co-founder of a company that uses the technology. The other authors declare that they have no conflicts of interest that could influence the contents of this article.

Data availability

No primary data was generated for this review article