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Relationship: 3633

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Increase, Protein oxidation leads to Decrease, Coupling of OXPHOS

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Increase, Protein oxidation

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

Decrease, Coupling of OXPHOS

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status
Excessive reactive oxygen species leading to growth inhibition via protein oxidation and cell injury/death	adjacent			Evgeniia Kazymova (send email)	Under development: Not open for comment. Do not cite
Reactive oxygen species leading to growth inhibition via protein oxidation and decreased cell proliferation	adjacent	Moderate	Low	Evgeniia Kazymova (send email)	Under development: Not open for comment. Do not cite
Reactive oxygen species leading to growth inhibition via protein oxidation and cell death	adjacent	Moderate	Low	Cataia Ives (send email)	Under development: Not open for comment. Do not cite

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Term	Scientific Term	Evidence	Link
humans	Homo sapiens	Moderate	NCBI
mammals	mammals	Moderate	NCBI
fish	fish	Moderate	NCBI
crustaceans	Daphnia magna	Moderate	NCBI
green algae	Ulva compressa	Moderate	NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Unspecific	Moderate

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
All life stages	Moderate

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

This KER describes the relationship by which increased protein oxidation leads to decreased coupling of oxidative phosphorylation. Protein oxidation refers to oxidative modification of protein amino acid residues or protein-associated cofactors, including carbonylation, thiol oxidation, methionine oxidation, tyrosine nitration, protein-peroxide formation, glutathionylation, and adduction by reactive aldehydes generated during lipid peroxidation. When such modifications affect mitochondrial proteins involved in electron transport, proton pumping, substrate transport, ATP synthase function, or maintenance of the inner mitochondrial membrane potential, OXPHOS efficiency can decline.

The downstream KE, decreased coupling of OXPHOS, describes a reduction in the efficiency with which electron transport and protonmotive force are coupled to ATP synthesis. AOP-Wiki Event 1446 describes this KE as dissipation or impairment of the protonmotive force across the inner mitochondrial membrane, measurable through decreased mitochondrial membrane potential, increased proton leak, altered oxygen consumption, or reduced respiratory control (AOP-Wiki, 2026c). Protein oxidation can contribute to this KE by impairing respiratory chain complexes, phosphate or nucleotide transporters, ATP synthase, redox cofactors, or mitochondrial membrane-associated proteins. This KER therefore links molecular damage to proteins with a cellular bioenergetic consequence.

The relationship is not intended to imply that all protein oxidation is adverse or that all oxidized proteins impair OXPHOS. Many reversible thiol modifications participate in redox regulation. The KER is most applicable when protein oxidation is persistent, extensive, affects mitochondrial or bioenergetic proteins, or exceeds cellular repair, reduction, and proteolytic capacity.

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

Evidence for this KER was assembled from the ROS-growth AOP network literature review and data-mining workflow, targeted searches of the primary literature, AOP-Wiki records, and mechanistic reviews of protein oxidation and mitochondrial bioenergetics. The evidence-collection process followed the AOP-Wiki KER page template, including structured consideration of taxonomic, life-stage and sex applicability; KER description; evidence supporting the KER; modulating factors; quantitative understanding; domain of applicability; and references.

Search concepts included combinations of the terms "protein oxidation", "protein carbonylation", "oxidized mitochondrial proteins", "thiol oxidation", "glutathionylation", "protein nitration", "oxidative damage", "mitochondrial dysfunction", "oxidative phosphorylation", "OXPHOS", "respiration", "complex I", "electron transport chain", "mitochondrial membrane potential", "proton leak", "respiratory control ratio", "ATP synthase", "hypoxia-reoxygenation", "cadmium", "hydrogen peroxide", "aging", "Daphnia", "Chlamydomonas", "bivalve", "fish", and "mammalian cell". Searches prioritized studies that measured upstream protein oxidation and downstream mitochondrial coupling or respiration endpoints in the same biological system or provided strong mechanistic evidence that oxidation of specific mitochondrial proteins impairs respiratory function.

AOP-helpFinder and preliminary text-mining were used to identify co-occurrence of event-related terms, followed by overlap analysis to remove redundant records and exclude low-priority literature. Large language model-assisted screening was used as an auxiliary tool to extract candidate metadata, identify evidence type, and prioritize abstracts and full-text records. Final inclusion, interpretation, and weight-of-evidence judgments were made by manual expert review. Mechanistic reviews were used to support biological plausibility, while primary studies were prioritized for empirical evidence and concordance assessment.

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

Biological plausibility of this KER is high. Proteins are major targets of oxidants because they are abundant, contain redox-active residues and cofactors, and often catalyze or participate in electron-transfer reactions. Reactive oxygen and nitrogen species, metal-catalyzed oxidants, lipid-derived aldehydes, and protein peroxides can modify cysteine, methionine, histidine, lysine, arginine, tyrosine and other residues, resulting in altered protein conformation, catalytic activity, complex assembly, stability, or degradation (Stadtman and Levine, 2003; Dalle-Donne et al., 2006; Davies, 2016).

Mitochondrial OXPHOS is particularly vulnerable to protein oxidation because it relies on multi-subunit protein complexes embedded in the inner mitochondrial membrane, iron-sulfur clusters, redox-active cofactors, substrate and nucleotide transporters, and maintenance of a protonmotive force. Oxidative modification of respiratory-chain subunits or transport proteins can reduce electron transfer, increase electron leak, impair proton pumping, alter substrate availability, or decrease membrane potential, thereby reducing coupling efficiency. Curtis et al. (2012) provided direct mechanistic evidence in 3T3-L1 adipocytes that increased carbonylation of mitochondrial proteins, including complex I-related proteins and transport proteins, was accompanied by decreased complex I activity, impaired respiration and reduced mitochondrial membrane potential. This provides a strong mechanistic bridge from the upstream KE to the downstream KE.

The relationship is also coherent with the broader OXPHOS AOP module. Decreased coupling of OXPHOS is a recognized measurable KE in AOP-Wiki and in the OECD-endorsed OXPHOS uncoupling leading to growth inhibition AOP. Although classical uncouplers act primarily through protonophoric mechanisms, oxidative damage to mitochondrial proteins provides an additional route to reduced coupling efficiency (AOP-Wiki, 2026c; OECD, 2022).

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

Empirical support for this KER is moderate. The strongest empirical evidence comes from studies in which increased mitochondrial protein carbonylation or oxidative protein damage is measured together with reduced mitochondrial membrane potential, impaired respiration, decreased complex activity, or reduced coupling efficiency. However, many studies report either protein oxidation or mitochondrial dysfunction without measuring both KEs in a manner that allows complete temporal, dose-response and incidence concordance assessment.

Evidence type	Summary of evidence	References
Direct mechanistic evidence in mammalian cells	GSTA4-silenced 3T3-L1 adipocytes displayed elevated carbonylation of mitochondrial proteins, including NADH dehydrogenase 1 alpha subcomplexes and phosphate carrier protein. Elevated protein carbonylation was accompanied by diminished complex I activity, impaired respiration, increased superoxide production and reduced mitochondrial membrane potential. Knockdown of selected carbonylation targets reduced basal and maximal respiration.	Curtis et al., 2012
Association in invertebrate life-history and mitochondrial function	Short-lived Daphnia pulex clones showed reduced complex I activity, increased oxidative damage and altered expression of ROS-scavenging enzymes. This supports an association between oxidative damage to cellular components and impaired mitochondrial respiratory function, although it does not isolate protein oxidation as the sole cause.	Ukhueduan et al., 2022
Bivalve hypoxia-reoxygenation evidence	Hypoxia-reoxygenation stress in the oyster Crassostrea gigas induced mitochondrial proteome and phosphoproteome shifts together with altered bioenergetic responses. This supports environmental relevance of oxidative/proteomic stress coupled to mitochondrial bioenergetic impairment, but direct protein oxidation-to-OXPHOS causality is not fully resolved.	Sokolov et al., 2019
Fish oxidative stress and mitochondrial response	Acute cold exposure in zebrafish brain induced oxidative stress responses and changes in uncoupling-protein/antioxidant mechanisms; protein carbonylation increased rapidly in the time course. This supports temporal feasibility of oxidative protein damage in relation to mitochondrial stress responses but does not provide a fully quantitative KER model.	Tseng et al., 2011
Photosynthetic eukaryote evidence	Large-scale redox proteomics in Chlamydomonas reinhardtii identified extensive protein glutathionylation under oxidative conditions, showing broad susceptibility of cellular proteins to redox modification. Evidence directly linking these modifications to decreased mitochondrial coupling in the same study is limited.	Zaffagnini et al., 2012

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

A key uncertainty is that protein oxidation is chemically diverse. Reversible thiol oxidation and glutathionylation can act as regulatory or protective modifications, whereas carbonylation, nitration, aggregation or irreversible oxidation are more likely to be associated with functional impairment. As a result, the biological consequence of the upstream KE depends strongly on the specific protein target, modification type, dose, duration and cellular context.

A second uncertainty is that decreased coupling of OXPHOS may result from multiple upstream mechanisms, including lipid peroxidation, direct chemical uncoupling, mitochondrial DNA damage, calcium dysregulation, permeability transition, complex inhibition, or changes in mitochondrial dynamics. Protein oxidation may be causal, contributory, or secondary to these other mechanisms. Empirical support is strongest when oxidative modification of mitochondrial proteins is measured together with respiratory endpoints, but such studies remain relatively limited across ecotoxicological species.

Temporal concordance may also be difficult to establish. Protein oxidation of susceptible residues can occur within minutes to hours, but detectable impairment of OXPHOS coupling may require accumulation of damage, modification of key targets, or failure of repair and proteolytic systems. Conversely, mitochondrial dysfunction can increase ROS production and promote further protein oxidation, creating a feedforward loop that complicates the assignment of a strictly unidirectional sequence.

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Modulating factor	Details	Influence on KER	Supporting evidence
Protein target and modification type	Carbonylation, nitration and irreversible oxidation of mitochondrial proteins are more likely to impair function than transient reversible thiol modifications.	Alters magnitude and probability of downstream OXPHOS impairment; oxidation of respiratory-chain subunits or transporters has higher expected impact.	Stadtman and Levine, 2003; Dalle-Donne et al., 2006; Davies, 2016; Curtis et al., 2012
Antioxidant and reductive repair capacity	Glutathione, thioredoxin, peroxiredoxins, methionine sulfoxide reductases and related systems can reverse or limit some oxidative protein modifications.	Higher antioxidant/repair capacity raises the threshold for downstream mitochondrial impairment.	Sies et al., 2017; Davies, 2016
Proteostasis capacity	Proteasomal and mitochondrial protein quality-control systems remove damaged proteins; reduced turnover permits accumulation.	Impaired proteostasis increases persistence of oxidized mitochondrial proteins and may increase downstream effect size.	Dalle-Donne et al., 2006; Davies, 2016
Mitochondrial abundance and energy demand	Cells with high mitochondrial density or high ATP demand may show stronger consequences of oxidation of OXPHOS proteins.	May increase sensitivity of downstream coupling endpoints to upstream protein oxidation.	Murphy, 2009; Nicholls and Ferguson, 2013
Exposure duration and intensity	Short transient oxidant pulses may cause reversible modification; persistent or high-intensity exposures can produce irreversible carbonylation and dysfunction.	Determines whether protein oxidation remains adaptive/regulatory or becomes damaging and functionally linked to OXPHOS impairment.	Davies, 2016; Curtis et al., 2012
Temperature, hypoxia-reoxygenation and oxygen availability	Environmental oxygen fluctuations and temperature stress affect ROS production, mitochondrial function and protein oxidation.	Can amplify oxidative modification and alter the timing and magnitude of downstream mitochondrial impairment.	Tseng et al., 2011; Sokolov et al., 2019

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

Quantitative understanding of this KER is low to moderate. The qualitative linkage between oxidative modification of mitochondrial proteins and impaired mitochondrial coupling is well supported, and individual studies provide quantitative data on protein carbonylation, complex I activity, oxygen consumption and mitochondrial membrane potential. However, there is currently no generalizable mathematical model that predicts the magnitude of decreased OXPHOS coupling from a given amount of total protein oxidation across taxa, tissues, stressors and measurement platforms.

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

The response-response relationship is expected to be nonlinear and target-dependent. Total protein carbonyls or other bulk oxidation markers may correlate poorly with OXPHOS impairment if oxidation occurs mainly in proteins unrelated to mitochondrial respiration. Conversely, relatively small amounts of oxidation affecting key respiratory-chain subunits, ATP synthase, inner membrane transporters, or proteins required for maintenance of mitochondrial membrane potential may have substantial bioenergetic consequences. Curtis et al. (2012) provide a strong example of response-response evidence because elevated carbonylation of specific mitochondrial proteins was accompanied by reduced complex I activity, altered oxygen consumption and reduced membrane potential.

Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

The time scale can range from minutes to days. Oxidation of susceptible mitochondrial protein residues may occur rapidly during an oxidant pulse, while measurable decreases in coupling efficiency may appear after sufficient oxidation of functionally important targets or after compensatory mechanisms are overwhelmed. In vivo studies of oxidative stress responses in fish show that protein oxidation can increase within hours under acute stress (Tseng et al., 2011), whereas environmentally relevant hypoxia-reoxygenation or chronic oxidative damage may alter mitochondrial proteome and function over longer time scales (Sokolov et al., 2019; Ukhueduan et al., 2022).

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

A biologically important feedforward loop may occur because impairment of mitochondrial OXPHOS can increase electron leak and ROS production, which can further oxidize mitochondrial proteins. This loop can amplify the KER once mitochondrial protein oxidation begins to impair electron transport or membrane coupling. Negative feedback or adaptive responses may include activation of antioxidant pathways, increased protein turnover, mitophagy, mitochondrial biogenesis, and metabolic compensation through glycolysis. These feedback mechanisms are expected to influence the threshold and persistence of the downstream KE but are not yet sufficiently quantified for general application.

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

This KER is most applicable to aerobic eukaryotic cells and tissues in which mitochondria are important for ATP production and in which protein oxidation affects proteins involved in mitochondrial respiration, membrane potential, substrate transport or ATP synthesis. It is applicable across a broad range of taxa because the underlying chemistry of protein oxidation and the core architecture of OXPHOS are conserved. Applicability is strongest when the upstream KE is measured using specific protein oxidation endpoints, such as protein carbonyls, oxidized thiols, nitrated proteins, methionine oxidation, glutathionylation or redox proteomics, and when the downstream KE is measured using mechanistically informative mitochondrial endpoints such as membrane potential, oxygen consumption rate, respiratory control ratio, proton leak, ATP-linked respiration, or complex activity.

Confidence is lower when protein oxidation is measured only as a nonspecific bulk endpoint, when mitochondrial dysfunction is measured only as general cytotoxicity, or when the two KEs are not measured in the same biological system. The KER should also be interpreted cautiously under conditions where direct chemical uncoupling, lipid peroxidation, mitochondrial DNA damage, or generalized cell injury may be the dominant cause of decreased OXPHOS coupling.

References

List of the literature that was cited for this KER description. More help

Almaida-Pagán PF, Lucas-Sánchez A, Tocher DR. 2014. Changes in mitochondrial membrane composition and oxidative status during rapid growth, maturation and aging in zebrafish, Danio rerio. Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids 1841(7):1003-1011. https://doi.org/10.1016/j.bbalip.2014.04.004.

AOP-Wiki. 2026a. Relationship 3633: Increase, Protein oxidation leads to Decrease, Coupling of OXPHOS. https://aopwiki.org/relationships/3633. Accessed 14 May 2026.

AOP-Wiki. 2026b. Event 1767: Increase, Protein oxidation. https://aopwiki.org/events/1767. Accessed 14 May 2026.

AOP-Wiki. 2026c. Event 1446: Decrease, Coupling of oxidative phosphorylation. https://aopwiki.org/events/1446. Accessed 14 May 2026.

Curtis JM, Hahn WS, Stone MD, Inda JJ, Droullard DJ, Kuzmicic JP, Donoghue MA, Long EK, Armien AG, Lavandero S, Arriaga E, Griffin TJ, Bernlohr DA. 2012. Protein carbonylation and adipocyte mitochondrial function. Journal of Biological Chemistry 287(39):32967-32980. https://doi.org/10.1074/jbc.M112.400663.

Dalle-Donne I, Aldini G, Carini M, Colombo R, Rossi R, Milzani A. 2006. Protein carbonylation, cellular dysfunction, and disease progression. Journal of Cellular and Molecular Medicine 10(2):389-406. https://doi.org/10.1111/j.1582-4934.2006.tb00407.x.

Davies MJ. 2016. Protein oxidation and peroxidation. Biochemical Journal 473(7):805-825. https://doi.org/10.1042/BJ20151227.

Murphy MP. 2009. How mitochondria produce reactive oxygen species. Biochemical Journal 417(1):1-13. https://doi.org/10.1042/BJ20081386.

Nicholls DG, Ferguson SJ. 2013. Bioenergetics 4. London: Academic Press.

OECD. 2022. Uncoupling of oxidative phosphorylation leading to growth inhibition via decreased cell proliferation. OECD Series on Adverse Outcome Pathways No. 28. Paris: OECD Publishing. https://doi.org/10.1787/f20867c1-en.

Perry SW, Norman JP, Barbieri J, Brown EB, Gelbard HA. 2011. Mitochondrial membrane potential probes and the proton gradient: a practical usage guide. BioTechniques 50(2):98-115. https://doi.org/10.2144/000113610.

Schieber M, Chandel NS. 2014. ROS function in redox signaling and oxidative stress. Current Biology 24(10):R453-R462. https://doi.org/10.1016/j.cub.2014.03.034.

Sies H, Berndt C, Jones DP. 2017. Oxidative stress. Annual Review of Biochemistry 86:715-748. https://doi.org/10.1146/annurev-biochem-061516-045037.

Sokolov EP, Markert S, Hinzke T, Hirschfeld C, Becher D, Ponsuksili S, Sokolova IM. 2019. Effects of hypoxia-reoxygenation stress on mitochondrial proteome and bioenergetics of the hypoxia-tolerant marine bivalve Crassostrea gigas. Journal of Proteomics 194:99-111. https://doi.org/10.1016/j.jprot.2018.12.009.

Stadtman ER, Levine RL. 2003. Free radical-mediated oxidation of free amino acids and amino acid residues in proteins. Amino Acids 25(3-4):207-218. https://doi.org/10.1007/s00726-003-0011-2.

Tseng YC, Chen RD, Lucassen M, Schmidt MM, Dringen R, Abele D, Hwang PP. 2011. Exploring uncoupling proteins and antioxidant mechanisms under acute cold exposure in brains of fish. PLoS ONE 6(3):e18180. https://doi.org/10.1371/journal.pone.0018180.

Ukhueduan B, Schumpert C, Kim E, Dudycha JL, Patel RC. 2022. Relationship between oxidative stress and lifespan in Daphnia pulex. Scientific Reports 12:2354. https://doi.org/10.1038/s41598-022-06279-4.

Zaffagnini M, Bedhomme M, Groni H, Marchand CH, Puppo C, Gontero B, Cassier-Chauvat C, Decottignies P, Lemaire SD. 2012. Glutathionylation in the photosynthetic model organism Chlamydomonas reinhardtii: a proteomic survey. Molecular & Cellular Proteomics 11(2):M111.014142. https://doi.org/10.1074/mcp.M111.014142.