Lipid-Oxidative Enzymes and Fenton-Like Reactions Are Synergistic in Promoting Membrane Lipid Peroxidation

Abstract

Lipid peroxidation (LPO) of cellular membranes is a near-universal indicator of aging and disease, yet the mechanistic link between the LPO and disease remains elusive. In this study, we demonstrate that efficient LPO in model membranes is accomplished through synergy between selective enzymatic oxidation by lipoxygenase (LOX) and nonspecific oxidation by reactive oxygen species (ROS). Through fluorescence-based oxidation kinetic measurements, we show that soluble ROS alone fails to induce significant oxidation under physiologically relevant conditions. However, enzymatic oxidation enhances the ROS-driven LPO by altering membrane permeability. Strikingly, this process drives the macroscopic clustering of the membrane-bound protein KRAS on giant unilamellar vesicles (GUVs), revealing potential functional consequences. If this mechanism extends to living cells, it could reshape our understanding of oxidative stress in disease. Our findings represent an essential step toward advancing an integrated understanding of oxidative membrane biology, encompassing both enzymatic oxidation and oxidation by ROS.

Introduction

Lipid peroxidation (LPO) of cellular membranes is a ubiquitous process that plays an important role in initiating and mediating the aging process. LPO is also associated with a legion of health concerns such as Alzheimer’s and Parkinson’s diseases, cancer, cardiovascular diseases, and numerous others. However, the connection between these pathologies and LPO reactions remains mysterious. Two prominent questions regarding the role of LPO in disease are as follows: − (1) what is the primary molecular mechanism responsible for membrane lipid oxidation, and (2) how does it lead to the pathogenesis of various ailments as well as the aging process? Answering these questions will enable a better understanding of the diseases whose pathology involves LPO and then potential strategies for effective treatments.

In LPO, carbon–carbon double bonds in lipids such as polyunsaturated fatty acids, phospholipids, glycolipids, and cholesterol undergo hydrogen abstraction and oxygen insertion in the allylic position, resulting in lipid peroxyl radicals and hydroperoxides (Figure ). This process may be initiated by enzymes such as lipoxygenases (LOX) as well as reactive oxygen species (ROS) generated from uncontrolled oxidative stress. In Fenton and Fenton-like reactions, labile metals such as Fe^III/II or Cu^II/I catalyze the reduction of H₂O₂, producing oxygen-centered radicals. Evidence in favor of the nonenzymatic reactions being the driving force of LPO includes the observation that iron chelators and radical trapping antioxidants inhibit the accumulation of LPO products. , In addition, an excess pool of labile iron leads to LPO. On the other hand, studies have found that LOX inhibitors protect cells from LPO, and the LPO products show specific oxygenation centers, supporting the enzymatic catalysis as the primary mechanism for membrane LPO. Furthermore, the regulated nature of LPO is thought to be fundamentally at odds with the nonspecific and uncontrolled iron-catalyzed radical chain reactions. In this study, we addressed these mechanistic questions regarding LPO using model membrane systems, eliminating the unknown complexities inherent in living cells.

1.

Two general pathways to lipid peroxidation in cell membranes. In enzymatic LPO, enzymes such as lipoxygenase (LOX) catalyze the peroxidation of lipids, particularly polyunsaturated fatty acids (PUFAs) such as linoleic acid and arachidonic acid. In nonenzymatic LPO, also known as autoxidation, reactive oxygen species (ROS) generate lipid radicals in a chain reaction, which is terminated through radical combinations or by radical-trapping antioxidants.

We hypothesized that both enzymatic and nonenzymatic reactions are important in LPO due to the complementary ways through which oxidation sites are accessed. This hypothesis was based on the well-established and experimentally supported observation that lipid peroxidation alters membrane structure in a manner that increases permeability, as demonstrated in multiple prior studies. , Since enzymes are capable of selectively oxidizing lipids in intact membranes, subsequent oxidation by ROS may become more effective in accessing the oxidation sites in the modified membranes. To test this hypothesis, we developed a fluorescence-based method to measure the reaction kinetics of LPO in liposomes. This approach enabled the quantification and comparison of LPO kinetics under various chemical conditions. Our major findings are as follows: (1) ROS generated from Fenton-like reactions such as hydroxy radicals (·OH) is inefficient in penetrating the tightly packed membrane and accessing acyl chains for oxidation, but (2) selective oxidation by an enzyme reduces the membrane packing so that ROS can enter and oxidize lipids, and (3) this can lead to significant chemical modification on membrane-bound proteins such as the KRAS. These results offer a new perspective on chemical mechanisms underlying LPO and LPO-associated diseases.

Results and Discussion

Measurement of LPO Kinetics in Membranes

We developed a method to quantify the oxidation kinetics of LPO in membranes, as described previously and depicted in Figure . This method utilizes enzymatic oxidation to trigger the LPO in liposomes. Large unilamellar vesicles (LUVs, ∼100 nm diameter) containing phosphatidylcholine (PC) such as 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), linoleic acid (LinA), and diphenylhexatriene (DPH) are prepared by extrusion (Figure A). This process produced monodispersed vesicles with an average diameter of 102 nm (Figure S1). In the first step of a two-step reaction (Figure B), soybean lipoxygenase-1 (LOX) catalyzes the oxidation of linoleic acid (LinA) to form linoleate hydroperoxide (LinAOOH). LOX has been confirmed to be active and to exhibit Michaelis–Menten kinetics without an additional activation step, with K _m and V _max values comparable to previously reported results. LinAOOH formation is detected by ultraviolet absorbance at 235 nm (Figure C).

2.

Measurement of membrane oxidation kinetics. (A) Large unilamellar vesicles (LUVs) composed of phosphatidylcholine (PC), linoleic acid (LinA), and diphenylhexatriene (DPH) probe are prepared. (B) Soybean lipoxygenase-1 (LOX) catalyzes the peroxidation of LinA, which then reacts with DPH. The generation of LinAOOH is measured by absorbance at 235 nm, and the oxidation of DPH is measured by fluorescence emission at 431 nm. In the kinetics measurements, a saturating concentration of LOX (1 μM) was used such that the oxidation of DPH follows a pseudo-first-order kinetics with the apparent reaction rate constant k′. (C) Time-dependent formation of LinAOOH by various LOX concentrations monitored by UV–vis. Membrane compositions were 10% LinA, 89.8% DOPC, 0.2% DPH. (D) Oxidation of DPH with 1 μM LOX is monitored by fluorescence at 431 nm. Membrane compositions were 0.2% DPH, various % LinA, and the rest DOPC. All experiments were performed in 150 mM tris-buffered saline (TBS), pH 7.4, at 37 °C.

Then, LinAOOH oxidizes the fluorescent probe DPH in the second step, which is monitored by a loss of fluorescence at 431 nm (Figure D). The second step is the reaction of interest that will reflect the overall oxidative environment of the membrane. To simplify the analysis, a high concentration (1 μM) of the enzyme is employed to complete the first reaction effectively instantaneously. The complete conversion to LinAOOH is established by its absorbance at 235 nm with the known extinction coefficient 23,600 ± 500 M^–1cm^–1 (Figure C). The second reaction then follows pseudo-first-order kinetics, with the rate constant k’. This method has been used to quantify the oxidation kinetics of various fluorescent sensors such as BODIPY-C11 and Liperfluo.

The apparent oxidation kinetics of DPH reflect the overall oxidative environment of the membrane. Figure shows the DPH oxidation kinetics in LUVs composed of PC of different degrees of saturation. DMPC is saturated, so it cannot become oxidized or oxidize DPH. However, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) and DOPC contain one and two double bonds, respectively, that may be oxidized at the allylic position and propagate oxidation. The apparent DPH oxidation kinetic rate constant k′ in each of the membranes increases with the degree of PC saturation (Figure A,B). The differences in oxidation kinetics could be due to the differences in the membrane diffusivity. However, at 37 °C, all PCs are in the fluid state, and a previous work has shown that the lipid peroxide-selective oxidation rate does not change between DMPC and DOPC under the same conditions. This suggests that indirect oxidation mediated by oxidized PC (for SOPC and DOPC) contributes to the overall DPH oxidation rather than increased lateral diffusion.

3.

Oxidation kinetics of DPH in different membranes. (A) The oxidation rate of DPH is dependent on the degree of PCs saturation (DMPC-0, SOPC-1, DOPC-2) in vesicles. Membrane compositions were 89.8% PC, 10% LinA, and 0.2% DPH. (B) Structure of DMPC, SOPC, and DOPC. (C,D) MALDI-TOF mass spectra and proposed reaction mechanisms for oxidized DOPC (C) and DOPC-cross-linked DPH (D). (E) Proposed oxidation reaction mechanism for DOPC and (F) cross-linking between DOPC and DPH.

To identify the reactions occurring during the LPO experiments, matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry was performed on the DOPC vesicle contents after oxidation. An oxidized DOPC product and cross-linked product between DPH and oxidized PC are found in the mass spectra (Figure C,D). Based on these results, we propose reaction mechanisms for the oxidation of DOPC and its cross-linking with DPH. The oxidation of DOPC to form an α,β-unsaturated carbonyl compound (800.58 m/z) is shown in (Figure E). Hydroperoxides such as LinAOOH are unstable and prone to decomposition into alkoxy and peroxyl radicals, which initiate LPO. The subsequent reaction steps align with established mechanisms of lipid peroxidation with the formation of the α,β-unsaturated carbonyl compound proceeding via a β-scission reaction. These findings indicate that LinAOOH, while acting as the primary oxidant, facilitates the formation of secondary reactive lipid species that propagate oxidation beyond its initial enzymatic targets. Given that SOPC and DOPC are not direct targets of LOX activity, their oxidation must be mediated by radicals, such as alkoxy and peroxyl radicals, generated from the homolytic decomposition of LinAOOH. Consequently, the indirect oxidation pathway manifests as the observed enhancement in DPH oxidation rates in unsaturated lipids.

The oxidative reactions involving phospholipid alkoxy radicals were further confirmed by the detection of a DPH-DOPC cross-linked product in the mass spectra at 1034.30 m/z (Figure D). The cross-linking is proposed to occur through an oxygen-mediated mechanism, where the double bond of DPH is connected to the allylic position of DOPC via radical cross-linking (Figure F). Peroxyl radicals are likely to play a critical role in generating the allylic radical from DOPC due to their comparatively greater stability (hence lower reactivity) and associated longer half-life (<7 s) relative to other radicals, such as alkoxyl (<10^–6 s) or hydroxyl (<10^–9 s) radicals.

ROS Alone Is Inefficient in Oxidizing Intact Membranes

Nonenzymatic LPO was observed by measuring the DPH oxidation kinetics in the presence of a Fenton-like reaction. Figure A shows DPH oxidation kinetics in DMPC and DOPC membranes exposed to H₂O₂ and Cu²⁺, which has been previously used for effective Fenton-like reaction in LPO. , Minimal oxidation of DPH and, by extension, oxidation of PC is observed up to 1 mM H₂O₂ and 7 μM Cu²⁺. Cytosolic H₂O₂ varies according to cell types but are typically regulated at 1–100 nM on average under normal conditions, reaching up to 1 μM during oxidative stress. 1 mM H₂O₂ and 7 μM Cu²⁺ thus represent oxidative conditions exceeding the average physiological levels, although there could be microenvironments with further elevated ROS concentrations. DPH oxidation is faster in DOPC than in DMPC, indicating that it is primarily mediated by oxidized PC rather than direct oxidation of DPH by ROS. Overall, these results suggest that under physiologically relevant conditions, ROS is ineffective in oxidizing lipids within intact membranes.

4.

(A) DPH oxidation kinetics by Fenton-like chemistry and LOX, at ROS levels up to and exceeding elevated physiological oxidative stress. Membrane compositions were 99.8% DMPC or DOPC and 0.2% DPH. (B) DPH oxidation with ROS (1 mM H₂O₂/7 μM CuSO₄), LOX (100 nM), and both, demonstrating a synergistic effect. Membrane composition was 89.8% DOPC, 10% LinA, and 0.2% DPH. (C,D) LUVs composed of DMPC (C) and DOPC (D) with various % LinA were oxidized by LOX (1 μM); then ROS (1 mM H₂O₂/7 μM CuSO₄) was introduced, and DPH oxidation kinetics were measured. (E) Comparison of DPH oxidation in various lipid compositions including DOPS (containing an anionic headgroup) and DLPC (containing bis-allylic fatty acid chains).

ROS Are Active on Enzymatically Oxidized Membranes

To test the hypothesis that both enzymatic and nonenzymatic oxidation is required for efficient LPO, their kinetics were measured individually and in combination (Figure B). Experiments were performed at excess ROS concentrations so that observations could be made within a 1 h time window. In vesicles composed of 89.8% DOPC, 10% LinA, and 0.2% DPH, enzymatic reactions were initiated by 0.1 μM LOX, and Fenton reaction was initiated with 1 mM H₂O₂/7 μM CuSO₄. There is a significant enhancement in the overall DPH oxidation rate when both are present that is greater than the sum of individual reactions (see Supporting Information for a statistical analysis). The catalytic rate of LOX itself is unaffected by Fenton reagents, as LinAOOH formation measured by absorbance at 231 nm does not change.

To examine the chemical origin of the synergistic effect between LOX and ROS in LPO, it was compared in DMPC and DOPC vesicles. Figures C,D show the DPH oxidation kinetic rate constant k’ in vesicles with varying % LinA in DMPC and DOPC, respectively. There is minimal oxidation by ROS alone and oxidation due to LOX increasing with the amount of its substrate LinA. The reaction rate is linear with % LinA in DMPC (Figure C), but it deviates from linearity in DOPC (Figure D) due to oxidized PC contributing to DPH oxidation. For the measurements of the synergistic effect (ROS + LOX), all LinA were first oxidized near-instantaneously with 1 μM LOX. After the enzymatic reaction was complete, ROS (1 mM H₂O₂/7 μM CuSO₄) was added at t = 0. The addition of 1 mM H₂O₂ deactivates LOX (Figure S3); however, as it no longer participates in LPO due to the depletion of LinA, the inactivation does not impact the measurement. The potential release of iron could also contribute to the overall LPO by ROS, although Fe³⁺ is expected to be much less productive of ROS than Cu²⁺ at neutral pH. The synergistic effect is dependent on % LinA, which means that it is dependent on the amount of preoxidized lipids. The effect is only significant in DOPC, suggesting that DPH oxidation is still primarily driven by oxidized PC rather than by direct oxidation of DPH by ROS.

Membrane Composition Can Tune LPO by ROS

There are lipids that can influence the oxidation rate by ROS. For example, phospholipids with anionic headgroups such as phosphatidylserine (PS) and phosphatidylethanolamine (PE) have been shown to chelate to divalent metal ions such as copper and calcium, bringing them close to the membrane and accelerating lipid oxidation. , DOPS is an abundant component in the inner leaflet of mammalian cells and contributes to the overall negative charge. Polyunsaturated phospholipids such as 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC) are especially vulnerable to oxidation, due to the low activation energy of bis-allylic hydrogen abstraction. Phospholipases liberate PUFAs from these lipids for subsequent oxidation by lipoxygenases, which can then act as signaling molecules in inflammation signaling.

Figure E illustrates DPH oxidation kinetics in LUVs containing PS and DLPC via enzymatic oxidation, ROS exposure, or both. The presence of 10% PS enables oxidation by ROS to a modest degree but does not significantly improve the cooperative effect. In contrast, substantial ROS-driven oxidation occurs with DLPC. At 10% DLPC and 90% DOPC, there is little DPH oxidation by LOX or ROS alone. When 10% linoleic acid and 10% DLPC are added to DOPC, enzymatic oxidation increases, aligning with the expectation that a higher degree of unsaturation enhances the oxidation rate. In addition, the enzymatic oxidation of LinA preceding ROS leads to the synergistic effect. Peroxides formed from DLPC are converted into DLPC-13-oxo-9,11-tridecadienoic acid residues and pentyl radicals. Because oxidized DLPC is unable to oxidize DOPC, as LinAOOH does, the oxidation rate in 10% DLPC is slower than that in 10% LinAOOH. Nevertheless, the presence of DLPC does not inhibit the synergistic effect.

Membrane Becomes Permeable to ROS after Enzymatic Oxidation

We investigated the diffusion of ROS across membranes when the lipids are intact or have been oxidized by LOX. It has been shown consistently that oxidized membranes have decreased viscosity and increased permeability, and computational work supports oxidized membranes becoming penetrable to ROS such as H₂O₂, ·OOH, and ·OH. Dichlorodihydrofluorescein (DCFH) is a commonly used ROS sensor that, upon reacting with ROS, emits green fluorescence (Figure A, also Figure S4). LUVs composed of LinA, DOPC, and Texas Red-DHPE and encapsulating DCFH were immobilized on the surface of a glass coverslip by using biotin–avidin conjugation. The vesicles were visualized by Texas Red fluorescence (l _ex = 561 nm) with total internal reflection fluorescence (TIRF) microscopy. In the presence of LOX or ROS individually, DCFH molecules inside the vesicles remain dark.

5.

Radical oxygen species cross-oxidized membranes. (A) DCFH is a ROS indicator that becomes fluorescent in green upon reacting with ROS. Lipid composition was 88.995 mol % DOPC, 1 mol % biotin-PE, 10 mol % LinA, 0.005 mol % Texas Red-DHPE. (B) LUVs encapsulating DCFH are immobilized on the glass surface by biotin–NeutrAvidin conjugation. Texas Red-DHPE indicates the location of individual LUVs (column 1). ROS does not cross the membrane when oxidized by LOX (column 2) or ROS (column 3) individually, but they diffuse through the membrane that has been oxidized by LOX and reacts with DCFH, resulting in green fluorescence within the vesicles (column 4).

However, when both LOX and ROS are present, there is green emission from the vesicles in the TIRF (l_ex = 488 nm). Only a subset (∼15%) of the vesicles becomes fluorescent, reflecting the encapsulation probability of DCFH in LUVs prepared by extrusion. The rise of the fluorescence signal, which is probably dictated by the diffusion of ROS across membranes, is very fast and occurs as soon as ROS is added to the imaging chamber. Despite the low encapsulation efficiency, the permeabilization effect was consistently observed across replicates and was highly specific to the LOX + ROS treatment (Figure S4). DCFH remains stably encapsulated in ∼102 nm LUVs during the 5 min TIRF measurement, even under oxidative stress, due to its relatively large size. The low responding fraction is therefore attributed to limited initial encapsulation, not loss during imaging. These vesicle-encapsulated ROS sensor results suggest that ROS are unable to diffuse through intact membranes on their ownbut can penetrate the barrier and further oxidize lipids, once the membrane has been broken by enzymatic peroxidation.

ROS-Mediated Oxidation of Membrane-Bound Protein KRAS

Our results provide strong precedent that the combination of enzymatic and nonenzymatic LPO impacts the oxidative susceptibility of model membranes. The implication is that oxidative damage can, in turn, impact radical-oxidation propagation to the signaling transduction machinery. Since many proteins important in signal propagation are functionally localized on the membrane surface, the clinical implication is that such chemical environments may contribute to lipid peroxidation-mediated diseases.

To investigate how membrane peroxidation may impact proteins on the surface, we examined a membrane-anchored protein on giant unilamellar vesicles (GUVs). KRAS is a key signaling protein that serves as a switch for multiple critical signaling pathways such as PI3K/AKT and MAPK/ERK. , It is localized to the membrane by a farnesyl anchor, and the membrane attachment is supplemented by the electrostatic interactions between the polylysine motif and anionic headgroup lipids such as PS. Native and fully processed KRAS fused to enhanced green fluorescent protein (eGFP), purified from insect cells, was allowed to bind GUVs composed of 57% DOPC, 10% DOPS, 30% cholesterol, 2.5% LinA, and 0.5% Texas Red-DHPE. Although 30% cholesterol may alter membrane fluidity and permeability, its inclusion was necessary to maintain vesicle stability and enable imaging of KRAS. Figure shows GUVs visualized by Texas Red-DPHE and KRAS by eGFP with spinning disc confocal fluorescence microscopy. eGFP-KRAS is distributed homogeneously on GUVs, and it is unaffected when LPO has been induced by LOX (1 μM) or ROS (1 mM H₂O₂/7 μM CuSO₄). However, when both are present, eGFP-KRAS appears to cluster on GUVs (see also Figures S5 and S6 and Movie S1). The clusters eventually dissociated from the GUVs. In three independent experiments, KRAS reliably formed clusters under this condition. However, the GUVs became highly unstable and ruptured shortly after KRAS clustering.

6.

eGFP-KRAS under oxidative stress on GUVs. GUVs are visualized by TR-DHPE (column 1). eGFP-KRAS spontaneously inserts into the membrane and is distributed homogeneously (column 2). The addition of LOX (column 3) or ROS (column 4) does not affect the uniform distribution. However, when both LOX and ROS are in the system (column 5), much of eGFP-KRAS leaves the membrane, and the remaining population forms visible clusters.

Mass spectrometry suggested that the clusters result from oxidative cross-linking of eGFP-KRAS. When eGFP-KRAS bound to LUVs was subjected to lipid peroxidation conditions (LOX combined with H₂O₂/Cu²⁺), MALDI-TOF revealed higher-molecular-weight species (Figure S6). These peaks were observed only under combined LOX and ROS treatment but not with either component alone. The eGFP-KRAS clustering could be due to oxidative cross-linking of any of its parts: the farnesyl anchor, the G-domain (the catalytic globular domain of KRAS), or eGFP. Farnesyl moieties may also be radicalized. However, it is unlikely to lead to the observed clustering, because if oxidative cross-linking occurs, it would be with much more abundant phospholipids in the membrane rather than another farnesyl anchor. Furthermore, the observation that the clusters eventually leave the membrane suggests that they are not cross-linked in the membrane. On the other hand, the G-domain of HRAS, which is 90% homologous in the amino acid sequence to KRAS, has been shown to be remarkably vulnerable to oxidative cross-linking by photosensitized oxidation. Specifically, dityrosines were formed when two tyrosine residues are radicalized and cross-linked, resulting in higher-order covalently linked clusters. Finally, eGFP has also been shown to be capable of becoming oxidatively cross-linked through dityrosine motifs without losing fluorescence. There are two possible mechanisms underlying the radicalization of the KRAS G-domain or eGFP upon LPO: (1) radicals are transferred from the lipids to proteins on the surface, or (2) ROS themselves react with the proteins as those ROS become concentrated near the membrane. In either case, oxidation originating from the membrane is expected to affect KRAS, which sits directly above the membrane, to a much greater extent than eGFP. These results show that the cooperative effect is likely to impact any proteins close to the membrane surface, potentially leading to the loss of function. ,

Conclusions

In this work, we identified synergism between enzymatic lipid oxidation and soluble hydroxyl radical species produced from Fenton-like reactions in membrane LPO and its chemically damaging impact on membrane-bound proteins. The oxidation kinetics measured with a fluorescent oxidation sensor, DPH, in model membrane vesicles indicated that soluble ROS generated by Fenton-like reactions are unable to react within the experimental time window of ∼ 1 h at 37 °C. However, if the membrane was selectively oxidized by lipoxygenase, then ROS was effective in propagating LPO. Membrane compositions such as the degree of unsaturation, charged headgroups, and bis-allylic groups may contribute to the extent of LPO. Single-vesicle imaging experiments demonstrated that ROS is only able to diffuse through the membrane when the membrane has been partially oxidized by LOX. This result suggests that the change in the membrane’s material properties allows ROS to access lipids for oxidation. This effect manifested dramatically on a membrane-bound protein, KRAS, which formed a macroscopic clustering on GUVs in the presence of both LOX and ROS.

A factor that may influence lipid peroxidation, but not experimentally examined here, is the molecular organization such as the one proposed by the lipid whisker model, in which the polar – OOH group of lipid hydroperoxides reorients toward the aqueous interface. However, surface exposure of hydroperoxides and increased membrane permeability are not mutually exclusive; both can occur concurrently and may synergize to amplify oxidative damage and facilitate interactions with membrane-associated proteins or reactive oxygen species. Another consideration not captured by this work is the potential oxidation of lipid headgroups by ROS, as the DPH fluorescence readout reflects the oxidation of the hydrophobic membrane core only. Finally, although not directly examined in this study, other reactive speciessuch as singlet oxygen, hypochlorite ions, reactive nitrogen species, and othersmay exhibit behavior similar to that of the ROS generated by Fenton-like reactions described here.

Because the results in this study were derived exclusively using model membranes, a remaining question for subsequent studies is to what degree does enzyme–ROS synergism play a role in the LPO of living cells. If the critical aspects of the chemical and material properties in in vitro membranes are conserved in living cellular membranes, then there is every expectation that our findings will prove more general. Indeed, the cooperative mechanism between lipid-oxidizing enzymes and ROS we have identified is one explanation for literature reports showing that inhibiting either ROS or lipid-oxidizing enzymes effectively reduces LPO. Furthermore, the KRAS cross-linking we see demonstrates that the membrane can transmit oxidative damage to membrane proteins through LPO, thereby establishing a mechanistic connection between LPO and disease. Overall, our study demonstrates that LPO displays emergent complexities, even at the most simplified level. It follows that any inhibition strategies of LPO should take these findings into consideration.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.5c00696.

• Experimental procedures and additional images to Figures 5 and 6 (PDF)

• A video showing KRAS clustering on GUVs upon LPO (MP4)

§.

H.J.J. and S.M. contributed equally to this work.

This work was supported by the National Institute of Health R35GM147482. H.J.J. was supported by the Basic Science Research Program through the National Research Foundation of Korea, the Ministry of Education (NRF-2021R1A6A3A14046029).

The authors declare no competing financial interest.