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. 2026 Mar 25;148(13):14443–14452. doi: 10.1021/jacs.6c01750

Origins of the Intrinsic Redox Activity of Biomolecular Condensates

Wen Yu †,§, Yanrun Zhou †, Leshan Yang †, Xiao Yan ‡, Samuel N Smukowski ∥, Yuefeng Ma †, Jiali Fan †, Young Ah Goo ∥, Anthony A Hyman ‡,*, Yifan Dai †,§,*
PMCID: PMC13067340  PMID: 41881426

Abstract

How inherent redox activity arises in biomolecular condensates remains unclear. Unlike interfacial systems, such as water microdroplets, where water oxidation underpins redox chemistry, condensates comprise biomolecules that can potentially furnish alternative electron-transfer routes. Here, using electron paramagnetic resonance, electrochemical potentiometry, mass spectrometry, and confocal microscopy assays, we discovered that orthogonal to water oxidation, microenvironment-dependent spontaneous tyrosine oxidation encodes an alternative redox pathway. Through proton-coupled electron transfer, self-induced tyrosine autoxidation in condensates drives the formation of reactive carbon and oxygen species, providing a pathway in parallel to hydroxide oxidation for hydrogen peroxide formation in condensates. This self-induced redox pathway modulates nonequilibrium condensate behaviors, including responses to external chemical perturbations and evolution of the condensate interior microenvironment. By correlating condensate biomolecular composition with inherent redox activities, our work establishes a conceptual framework suggesting that condensate-dependent electron transfer can be critical to define the functions of condensates and deliver a new redox mechanism for cell biology.

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Introduction

An emerging feature of biomolecular condensates is their inherent chemical activity, which is not encoded by specific biomolecular constituents but by the mesoscale electrochemical environments defined by the coexisting phases. These inherent chemical activities include mediating the aldol reaction, − the formation of hydrogen peroxide, − and catalyzing the hydrolysis reaction. A key driver underlying these activities is the interfacial field dependent redox activity of condensates, in which high-energy radicals and electrons are generated to promote diverse chemistry. ,− These previous works have been largely inspired by the studies of microdroplet water chemistry and “on water” catalysis, ,− in which the oxidation of hydroxyl ions and water structure-dependent charge transfer serve as the primary source driving the redox activities. ,,− This oxidation is mediated by the shift of free energy landscapes of hydroxyl ions and hydroxyl radicals at the interface relative to them at bulk, ,,− , rendering their oxidation to be thermodynamically favorable.

Compared to water microdroplets, ,,,− biomolecular condensates possess a highly complex molecular composition, − such as intrinsically disordered proteins (IDPs) and nucleic acids, ,, and unique microenvironments, such as distinct interior polarity, interfacial fields, ionic conditions, and pH. ,,− We posit that under the unique condensate microenvironment, the biomolecules within condensates can potentially introduce residues that can be activated to drive electron transfer. A critical residue in biomolecular systems that can contribute to electron transfer is tyrosine (Tyr), which is also highly preserved in IDPs, serving as a key “sticker” element in driving phase separation. − Tyrosine exploits its phenolic group (YOH) to drive proton-coupled electron transfer to form (YO•) and hydrogen radical (•H), suggesting that the oxidation potential of Tyr is coupled with pH condition, in which a decrease in proton content enhances the propensity to drive electron transfer. This environmental property favorable for tyrosine-dependent electron transfer also aligns with the alkaline interior of many condensates. , Further, local protein interactions, such as cation–pi interactions, which are abundant in condensates, , can reorganize the electronic distribution within the aromatic ring, thereby decreasing the oxidation potential of tyrosine. Such a polarization effect can likewise be enhanced by interfacial fields, which is another defining property driving condensate electrochemistry. , These features inspire us to study whether and how condensate-dependent tyrosine redox activity contributes to the inherent electrochemical activities of condensates.

Hydroxyl Radical Generated from Condensate-Dependent Water Oxidation

To study the molecular origin of the redox activities of condensates, we employed the resilin-like polypeptide (RLP: Ser-Ser-Gly-Pro-[Gly-Arg-Gly-Asp-Ser-Pro-Tyr-Ser]20-Gly-Tyr) to form condensates, which possess a basic interior environment and active interfacial field and are known to drive diverse chemical reactions, ,,, including generation of hydrogen peroxide, though the exact source of redox origin has not been directly studied. To this end, we first implemented electron paramagnetic resonance (EPR) spectroscopy to map the types of radicals generated in the RLP condensate solution. For these experiments, to exclude the effects of protein concentration, we prepared the “without condensate condition” using the same IDP concentration as the “with condensate condition” by introducing additional salt into the dilution buffer, which prevents condensate formation (Figure A). We confirmed that the added salt did not affect EPR analysis. Using 5,5-dimethyl-1-pyrroline N-oxide (DMPO), which is a widely applied radical trap for hydroxyl and carbon-centered radicals, we identified the characteristic quadruplet peaks (αN = 1.49 mT, αβ H = 1.49 mT), which is representative of the DMPO–OH adduct, confirming the existence of hydroxyl radical in the condensate system (Figure B). As a control to exclude contributions from potential trace metal contaminants from the water source, we added ethylenediaminetetraacetic acid (EDTA), a metal-chelating agent, to the condensate solution. Radical generation persisted in the presence of EDTA (Figure S1A), indicating that the observed oxidation does not arise from metal-catalyzed reactions in the bulk solution. Orthogonally, we applied inductively coupled plasma mass spectrometry to examine the water source (Table S1) and did not observe the noticeable presence of iron. We next reasoned that to fully exclude the possibility of the bulk redox reaction, which should originate from solvated oxygen, we applied H2 18O to prepare the dilution buffer so that if we can capture a hydroxyl radical containing 18O, this can further verify the water oxidation ability of condensates. Using (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-yl)­oxidanyl as the hydroxyl radical capturing agent, through mass spectroscopy, we confirmed the existence of R-18OH in a condensate solution (Figure S1B). These results collectively confirm that condensate can directly mediate water oxidation.

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Condensate-dependent generation of distinct reactive radicals. (A) Formation of protein condensates by dilution. Intrinsically disordered proteins (IDPs) undergo liquid–liquid phase separation, transitioning from dominant protein–solvent interactions to protein–protein interactions and producing micron-scale condensates. (B) Electron paramagnetic resonance (EPR) spectra of hydroxyl and carbon-centered radicals. Radical species were detected via spin-trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in solutions with or without condensates. Only condensate-containing samples exhibited the characteristic four-line hyperfine splitting of the DMPO–•OH adduct and the six-line splitting of the DMPO–•C adduct. Time-dependent EPR signal intensity of hydroxyl radical and carbon-centered radical in condensate-containing solutions. The EPR simulation corresponds to the EPR results of condensates formed after 20 min of incubation. (C) Time-dependent signals of carbon-centered and hydroxyl radicals in condensates, followed by measurements in condensate solutions treated with the hydroxyl-radical scavenger tert-butanol. (D) Effect of oxygen and hydroxyl radical scavenger tert-butanol on dityrosine formation. Dityrosine formation in RLPWT condensates formed under oxygen-minimized conditions compared with ambient condition. (E) EPR detection of superoxide radical. O2 •– generation was monitored using 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) as the spin probe.

Formation of Carbon-Centered Radical Independent of Hydroxyl Radical Formation

Interestingly, during EPR experiments, with increasing time of condensate formation, we observed the emergence of a typical hyperfine splitting spectrum of DMPO-R adduct (αN = 1.57 mT, αβ H = 2.36 mT) representing carbon-centered radical (Figure B). The formation of this radical is independent of the buffer we used (Figure S1C). Even for condensates formed in ultrapure molecular biology-grade water (with a pH at 5.8) without any buffering agent, we still observed the formation of this radical, suggesting that the source of this radical is the protein itself and its formation might be activated by the favorable microenvironment of condensates. We reasoned that based on our low-complexity protein sequence, this carbon-centered radical is highly possible to be originated from the oxidation of tyrosine, which leads to the formation of tyrosyl phenolic radical, and the delocalized electron transfers to the carbon on the aromatic ring, which eventually reacts with DMPO to form a stable adduct. , Our observations and simulated results align with previous works studying the carbon-centered radical in proteins using the DMPO assay. − This suggests that condensate-dependent oxidation of amino acids might occur in the condensate system. This observation raises two possibilities: (1) the formed hydroxyl radical further oxidizes the amino acid on protein chains, and (2) the condensate microenvironment powers the spontaneous oxidation of amino acids. The first possibility is unlikely, because hydroxyl radicals generated at the interface would be expected to affect only protein residues located at the condensate surface, due to the short self-diffusion length scale of hydroxyl radicals in aqueous solution. Experimentally, to evaluate this, we examined the correlation between time-dependent signals of carbon-centered radical and hydroxyl radical and included a control condition in which condensate solution was treated with hydroxyl radical scavenger, tert-butanol (Figures C and S2). We found that the addition of the hydroxyl radical scavenger decreased the EPR signal of the hydroxyl radical, while the time-dependent growth of the carbon-centered radical was unaffected. This observation suggests that the two distinct radicals were generated by orthogonal pathways.

Tyrosine Undergoes Autoxidation in Condensates

To evaluate whether the formed carbon-centered radical is tyrosine explicitly, we performed LC–MS/MS-based proteomics analysis to directly track the formation of DMPO-trapped tyrosine adducts under the same reaction conditions used for the EPR experiments. Using stringent site-localization filtering (Class I localization probability Q ≥ 0.75), we tracked the DMPO modifications on trypsin-digested peptide fragments and detected DMPO–Tyr modification on peptides within the RLPWT sequence only under the condition with condensates (Figure S3A). To evaluate whether oxygen might participate in condensate-dependent oxidation, we compared the DMPO modification using samples prepared in ambient and anaerobic conditions and found that the formation of the DMPO-Tyr product is independent of the existence of environmental oxygen, which aligns with our EPR observation.

In a parallel experiment, we reasoned that the formation of tyrosyl radical would eventually lead to the formation of dityrosine cross-links within condensates. To this end, we measured the absorbance of dityrosine using its unique UV–vis spectrum, and identified that the formation of dityrosine strictly depends on the existence of condensates and is independent of the presence of oxygen or hydroxyl radicals brought about by condensates (Figure D). These observations collectively confirmed that condensate formation can drive the autoxidation of tyrosine.

Transferred Electron Results in Oxygen Reduction

The generation of these radicals in condensate solution suggests the presence of solvated electrons. ,,− To this end, we used EPR with TEMPO, which is an electron trapping agent and confirmed the existence of solvated electrons in the condensate system (Figure S3B). We next reasoned that the electron would transfer to the solute species, such as solvated oxygen. To evaluate this, we applied 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH), which is a spin trap specific to superoxide radical (O2 •–). We found a time-dependent increase of the O2 •– (triplet peak) (Figure E). The superoxide signal gradually plateaued after 20 min. As a control to exclude contributions from trace metal contaminants in the water, EDTA was added to the condensate solution, and O2 •– formation persisted (Figure S1A). By purging the buffer with nitrogen to remove the dissolved oxygen, we confirmed that the generation of O2 •– is originated from dissolved molecular oxygen (Figure S3C).

To further confirm the existence of O2 •– in condensate solution, we applied the PO-1 assay, a fluorogenic probe specific to H2O2, with superoxide dismutase (SOD), an enzyme that can specifically catalyze the dismutation of O2 •– into H2O2. Compared to a solution with condensates but without SOD, a solution with condensates and SOD showed a significantly higher level of H2O2 signal (Figure S3D). This experiment orthogonally confirmed the existence of O2 •– through a biochemical approach. Further, by using dimethyl sulfoxide as the electron scavenger, through the same PO-1 assay, we found a substantially decreased amount of O2 •–, suggesting that solvated oxygen serves as an important electron acceptor in the condensate solution (Figure S3E). Collectively, these observations indicate that condensate redox activity can be driven by diverse, mechanistically distinct oxidizing sources.

Decreased Oxidation Potential of Tyrosine in Condensates

As the only residue in our sequence that is susceptible to oxidation under the experimental condition is Tyr, we next set out to study whether the condensate microenvironment, which encodes an alkaline pH for RLP condensates, can promote the oxidation of Tyr (Figure A). As Tyr presents a distinct electrochemical oxidation peak (Figure S4A), we employed electrochemical potentiometry to directly probe the change of oxidation potentials of Tyr in the subsaturated solution and the separated dense phase (Figure B). Through cyclic voltammetry scan, we observed a substantial decrease of the oxidation peak potential by approximately 50 mV in the dense phase compared to that of the subsaturated solution.

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Condensate modulates the oxidation potential of tyrosine. (A) Tyrosine undergoes proton-coupled electron transfer for oxidation. Phase separation generates an asymmetric ion distribution and a distinct electrochemical microenvironment. Elevated pH within condensates can facilitate tyrosine oxidation. (B) Oxidation potentials of tyrosine measured in the presence or absence of condensates. After bulk phase separation by high-speed centrifugation, cyclic voltammetry was performed to measure the tyrosine oxidation potential. A three-electrode system was utilized for electrochemical studies. A glassy carbon electrode, Ag/AgCl (KCl saturated), and a platinum disk electrode were used as the working, reference, and counter electrodes, respectively. (C) Cysteine-tethered peptide was fixed on an Au electrode surface by a Au–S bond. The addition of RLPWT promotes homotypic interactions with the tethered peptide, driving phase separation on the electrode. The resulting film was used to evaluate the effect of protein phase separation on the oxidation potential of Tyr. (D) Oxidation potentials of soluble RLPWT as a function of pH. (E) IDP phase separation generates a microenvironment in which elevated local pH and interfacial electric fields together enable tyrosine oxidation and intrinsic redox activity.

To verify this dense phase-dependent behavior, as an orthogonal strategy, we formed a dense 2D protein layer on the working electrode by introducing a tethering peptide that serves as the seed to form a protein layer (see Methods). This strategy is inspired by a recent work studying the redox chemistry of coacervates. We found that compared to the surface that only contains the tethering peptide, the formation of a dense protein layer on the surface also led to decreased oxidation potential of Tyr by approximately 43 mV in the RLP film (Figure C). These results suggest that condensates encode a microenvironment that can facilitate Tyr oxidation.

We next evaluated the oxidation potentials of subsaturated RLP in bulk solution under different pH conditions (Figure D), which showed that an alkaline environment decreases its oxidation potential. These results suggest that the alkaline pH environment of RLP condensates can substantially promote the activation of tyrosine-dependent proton-coupled electron transfer.

To verify this pH-dependent effect on Tyr oxidation, we next compared the oxidation behaviors of Tyr with those of phenylalanine (Phe), which does not possess the phenolic group responsible for electron transfer (Figure S4A). The disappearance of the oxidation peak suggests that the phenolic group on the aromatic ring is responsible for the electron transfer process. Further, by analyzing the pH-dependent oxidation potential of Tyr and Phe, we found that an alkaline pH, which corresponds to the interior pH of condensates, promotes tyrosine oxidation, as evidenced by a decreased current peak representing the quantity of Tyr monomer (Figure S4A). With further increasing pH until 11, we observed not only decreased oxidation potential but also gradual disappearance of the oxidation peaks, suggesting that Tyr oxidation occurs spontaneously under alkaline environments.

To verify the spontaneous oxidation of tyrosine, we applied UV–vis spectroscopy, which revealed the emergence of the characteristic dityrosine absorbance under alkaline conditions for both the tyrosine monomer and RLP in bulk solution (Figure S4B,C). These results suggest that the favorable interior pH encoded by condensates can lower the potential required for the oxidation of Tyr, which might be further facilitated by the interfacial field, thereby contributing to the inherent redox activity (Figure E).

Spontaneous Tyrosine Oxidation Contributes to the Formation of Hydrogen Peroxide

To understand the role of the phenolic group of Tyr in driving redox reactions, we designed a mutant RLP based on the same repeat number by replacing 10 of the repeat units [GRGDSPYS] with [GRGDSPFS], generating a Y7F mutant (SSGP-[GRGDSPFS]5-[GRGDSPYS]10-[GRGDSPFS]5-GY), in which 10 Tyr is replaced with 10 Phe (Figure A). We noticed that further decreasing the number of Tyr abolished condensate formation, so we selected this mutant for subsequent studies. Using the DMPO assay with EPR, we still identified the existence of hydroxyl radical (Figure B), suggesting that the oxidation of hydroxyl ions is an independent pathway contributing to the redox activity. This observation aligns with a recent study that demonstrates that condensates without tyrosine could still generate hydroxyl radicals. Notably, compared to RLPWT, we found that the signal generated from the carbon-centered radical largely disappeared in condensates formed by the Y7F mutant within the same testing period, which further confirms that Tyr is the residue mediating the formation of the carbon-centered radical. Using the CPH assay to analyze the O2 •–, we also found a substantial decrease of the intensity of the O2 •– signal (Figure C). By correlating the signal generated from O2 •– with •OH or carbon-centered radical (Figure D) and compared the same signal correlation factor from RLPWT condensates, we found that the correlation factor of •OH/O2 •– in the Y7F condensates substantially increased, suggesting that in the Y7F condensates, the formation of O2 •– majorly comes from the oxidation of hydroxyl ions due to the decrease of the tyrosine contents.

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Tyrosine-dependent generation of superoxide radical contributes to hydrogen peroxide production. (A) Schematic of the RLPY7F mutant used to probe the role of tyrosine’s phenolic group in redox reactions. (B) EPR spectra of RLPY7F condensates showing hydroxyl and carbon-centered radicals detected by spin-trapping with DMPO. Time-dependent EPR signal intensity of carbon-centered radicals in solutions containing condensates, comparing RLPWT condensates and RLPY7F condensates. The EPR simulation corresponds to condensates formed after 10 min of incubation. (C) EPR detection of superoxide (O2 •–) in RLPY7F condensates using CPH as the spin probe. Condensates exhibited the characteristic three-line hyperfine splitting of the CP• radical formed upon oxidation of CPH by O2 •–. (D) Kinetic correlation analysis of radical species in RLPWT and RLPY7F condensates. The time-dependent formation rates (k) of •OH or carbon-centered radicals were quantified and normalized by the corresponding O2 •– formation rate (k radical/k O2 •–). (E) Schematic illustration of the dual-mechanism pathway for H2O2 generation at condensate interfaces. The interfacial electric double layer concentrates ions and enhances local electric fields, while pH gradients within and around the condensates modulate tyrosine protonation states. Together, these effects promote tyrosine oxidation and redox reactions, ultimately leading to the formation of H2O2 at the interface. (F) Detection of H2O2 using the PO-1 assay. Conversion of PO-1 from its nonfluorescent to fluorescent form was quantified at 30 and 60 min in RLPWT and RLPY7F condensates. The contribution of superoxide to H2O2 formation was assessed by the addition of superoxide scavenger (p-benzoquinone) and •OH scavenger (tert-butanol) as separate treatments. Data represent mean ± s.d.; n > 20 condensates. Scale bar, 10 μm.

Lastly, using the PO-1 assay to quantify H2O2 production in the presence of different radical scavengers, we found that both O2 •– and •OH are critical to the formation of H2O2 by condensates (Figure F and see Discussion section for mechanistic details). The above observations reveal two distinct pathways for condensates to drive the formation of hydrogen peroxide (Figure E): (1) the spontaneous oxidation of tyrosine leads to the transfer of electrons to solvated oxygen to form superoxide, which undergoes dismutation to form hydrogen peroxide; (2) the spontaneous oxidation of hydroxy ions leads to (a) the transfer of electrons to form superoxide, which undergoes dismutation to form hydrogen peroxide, and (b) the recombination of hydroxyl radical to form hydrogen peroxide.

Spontaneous Tyrosine Oxidation Determines Condensate Stability and Microenvironment

We next wondered whether the condensate-mediated Tyr oxidation could modulate the condensate itself (Figure A). We reasoned that the condensate-dependent formation of di-Tyr cross-links could contribute to condensate stability (Figure B) because dityrosine formation is covalent cross-linking, which should mediate higher resistance to noncovalent perturbations. To this end, we designed a dissolution assay by titrating adenosine triphosphate (ATP), a hydrotrope that can disrupt electrostatic interactions, or 1,6-hexanediol, a molecule that can disrupt hydrophobic interactions, to compare the stability of condensates formed by RLPWT and the Y7F mutant. Apparently, the existence of Tyr in the condensate encodes a higher stability of condensates.

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Condensate-dependent redox activities modulate condensate stability and microenvironments. (A) Dityrosine formation generates interprotein cross-links that modulate condensate properties. (B) Interprotein dityrosine cross-linking enhances condensate stability. Dissolution analysis of RLPWT and RLPY7F condensates. Stability was assessed by titrating adenosine triphosphate (ATP) and 1,6-hexanediol (1,6-HEX), which selectively disrupt electrostatic and hydrophobic interactions, respectively. The dashed box marks the concentration range at which condensates dissolve. Scale bars, 10 μm. (C) Oxidation of tyrosine results in the reduction of molecular oxygen to superoxide (O2 •–), which subsequently forms hydrogen peroxide upon protonation. This consumption of protons makes the condensates more basic. (D) Representative C-SNARF-4 ratiometric images of RLPWT condensates and RLPY7F condensates in air-saturated and nitrogen-purged environments at defined time points. Data represent mean ± s.d.; n > 17 condensates. Scale bar, 1 μm. Oxygen removal stabilizes the pH change in condensates, indicating that oxygen not only contributes to the formation of hydrogen peroxide but also affects the internal environment of the condensates.

To understand whether this feature is linked with the oxidation capability of Tyr, we added a reductant molecule, dithiothreitol (DTT), to the system and tested the stability of condensates using the same dissolution assay (Figure S5). We found that with the presence of DTT in the solution system, the stability threshold of condensates under the treatments of the small molecules was substantially changed. We also verified the redox-dependent condensate environment using the Thioflavin T assay (Figure S6), which suggests that the condensate maintains a more hydrophobic environment in the absence of reductant. These results suggest that the inherent redox activities of condensates could contribute to condensate stability and microenvironments.

From the nonequilibrium aspect of condensates, we next wondered whether the condensate microenvironment could be affected due to tyrosine-dependent electron transfer. We reasoned that the one-electron oxidation of tyrosine leads to the formation of a superoxide radical, which in turn leads to the formation of hydrogen peroxide with the consumption of local protons (Figure C). Therefore, we wondered whether the consumed proton could result in a change in the condensate pH. To this end, we measured the time-dependent condensate pH using the C-SNARF-4 assay (Figure D). For RLP condensates, under normal experimental conditions, the condensate interior apparent pH gradually increased by approximately 0.5 pH units within 80 min. In contrast, when condensates were prepared in nitrogen-purged buffer, the interior apparent pH remained nearly constant. Further, by evaluating the time-dependent interior apparent pH of Y7F condensates, we found limited pH variation within the aging period (Figure D). These observations suggest that the condensate-dependent autoxidation of tyrosine modulates the nonequilibrium dynamics of condensate properties.

Discussion

Redox activities have been observed in different types of condensates; ,,,, however, the molecular origin has been largely unknown. Gleaned from the theories of microdroplet chemistry, where interfacial electric fields at liquid–liquid, liquid–air, and liquid–solid boundaries oxidize hydroxide to drive electron transfer and generate hydrogen peroxide, ,,,, past studies of condensate redox chemistry have assumed that condensates engage the same water-dependent oxidation pathway. ,,, However, the molecular complexity of biomolecular condensates far surpasses that of water microdroplets. This inspired us to uncover whether the biomolecular constituents could drive electron transfer.

A key redox center in biochemistry is tyrosine, the oxidation of which mediates the electron transfer in diverse cellular processes, including photosystem II, ribonucleotide reductase. The oxidation of tyrosine is a result of the migration of electron density to the phenolic oxygen. So, a favorable environmental pH, hydrogen bonding to the phenolic group, and a delocalization of electrons through pi-based interactions on the aromatic groups can all modulate the redox potential of tyrosine. The model condensate in this work, with an alkaline environment, facilitates the removal of the proton, thereby lowering the redox potential of tyrosine. Further, in condensate dense phase, many of the interactions are mediated by cation–pi interactions, which in our system is manifested by the Arg–Tyr interaction; such interactions can further stabilize the electron on the tyrosine, thereby lowering its redox potential. With the assistance of the interfacial field, such a barrier could further be lowered, thereby facilitating tyrosine oxidation. These findings suggest that condensates can potentially utilize their own microenvironments and dense phase interactions to modulate electron transfer.

From a biological aspect, tyrosine serves as the key interacting element in many phase-separating domains, such as FUS and hnRNP A1. − Recent works showed that stress granules can generate hydrogen peroxide spontaneously, which contributes to the formation of disulfide bonds of TDP-43 to promote its aggregation at the interface of stress granules-a key feature in neurodegenerative disorders. Also, considering the concentration effect by Tyr vs hydroxyl ions in the condensates, the higher abundance of protein concentrations (∼10–3 M) in the dense phase compared with hydroxyl ions (∼10–5–10–4 M) in some condensates , suggests that amino acids may play a more dominant role in driving electron transfer. We note, however, that the interfacial concentration of these species and the exchange kinetics of proteins between the dilute and dense phases may be the primary determinants of the rate of this process. Future work on uncovering the redox activity of tyrosine in the condensate microenvironment might expand our understanding of the redox activity of condensates and develop a novel therapeutic concept to prevent aggregation.

Our finding that autoxidation of tyrosine can further tune the proton contents of condensates further suggests that the inherent chemical activity of condensates is tightly coupled with the evolution of condensate microenvironments. Considering the great importance of condensate pH environments on modulating their cellular functions, , future studies might further uncover how the thermodynamic equilibrium of condensates is linked with the kinetics of their inherent chemical reactions. Recent advances in the de novo design of short peptides for phase separation could provide a simple and clean platform to establish the sequence-property relationship of these condensate-dependent redox features.

Although condensates appear to host highly oxidizing environments, any oxidation reaction is coupled to a reduction reaction. Thermodynamically, under the assumption of a bulk homogeneous environment, the electron should go to the species with the highest reduction potential in an aqueous environment. However, in a condensate system, which is defined by electrochemical potential equilibrium, the partitioning of chemical species is first dictated by the electrochemical potential gradients between two phases. So that the reduction reaction is collectively governed by the types of available chemicals, their concentration in the system, and their spatial distribution, which further affect the kinetics of the reaction. Such Janus chemical processes should be encoded in local chemical environments of condensates, suggesting that condensates in distinct cellular contexts can support distinct redox pathways. Uncovering these features in condensates might introduce a new paradigm for redox biology.

Supplementary Material

ja6c01750_si_001.pdf (4.9MB, pdf)

Acknowledgments

We appreciate experimental support from the Center for Biomolecular Condensates at Washington University in St. Louis.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.6c01750.

  • Experimental details, including plasmid construction and strain information, protein expression and purification, condensate preparation, radical detection, electrochemical characterization, and confocal fluorescence imaging assays (PDF)

⊥.

W.Y., Y.Z., and L.Y. These authors contribute equally.

Y.D. acknowledges the funding support from Alzheimer’s Association (AARG-25-1486936). A.A.H. acknowledge the funding support from Körber-Stiftung (Germany), the Max Planck Society (Germany), and the NOMIS Foundation (Switzerland). Mass spectrometry analyses were performed by the Mass Spectrometry Technology Access Center at the McDonnell Genome Institute (MTAC@MGI) at Washington University School of Medicine, supported by the Diabetes Research Center/NIH grant P30 DK020579, Institute of Clinical and Translational Sciences/NCATS CTSA award UL1 TR002345, and Siteman Cancer Center/NCI CCSG grant P30 CA091842.

The authors declare no competing financial interest.

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