Peptide‐Metal Cation Coacervate Microdroplets as Membrane‐free Protocells with Enhanced Light‐Induced Catalysis

ABSTRACT

Membrane‐free coacervates, typically formed through electrostatic interactions between oppositely charged polyelectrolytes, have been extensively utilized as artificial life‐like systems in protocell research, providing valuable insights into the origin of life. However, considering that inorganic metal ions may have been more abundant under early Earth conditions, it is plausible that these metal ions played a significant role in prebiotic compartmentalization. In this study, we present a novel coacervate protocell formed via liquid‐liquid phase separation (LLPS) of simple anionic polypeptides, induced by electrostatic and coordination interactions with inorganic metal cations, using divalent zinc ions as a specific example due to their prebiotic availability and biological relevance. The peptide‐zinc ion coacervate microdroplet exhibits liquid‐like properties, client partitioning, and enhanced catalysis comparable to those of typical polyelectrolyte coacervates. By integrating experimental results and theoretical simulations, we identify two kinetic pathways to phase separation mediated by the coordination mode at varying zinc ion concentrations, resulting in the formation of nonequilibrium gel‐like condensates or droplets that further contribute to distinct light‐induced catalytic efficiency. Overall, our work highlights that peptide‐metal ion interaction‐driven compartmentalization, as prebiotic microreactors, could have facilitated primitive biochemical reactions on early Earth, thus offering a plausible and diverse pathway for the emergence of protocells.

A novel peptide‐metal interaction‐based coacervate protocell, exhibiting liquid‐like properties, client recruitment capabilities, and enhanced enzymatic catalysis, facilitates biochemical processes mediated by coordination modes through the modulation of zinc ion concentrations, resulting in nonequilibrium gel‐like condensates or droplets.

1. Introduction

Compartmentalization is a fundamental pillar of living systems and represents a crucial step in the emergence of contemporary life [1, 2]. To understand how abiotic components can be organized into life‐like compartments capable of adaptation and evolution, various types of artificial microsystems for protocellular platforms have been developed [3, 4, 5, 6, 7, 8, 9, 10]. Among the existing cell‐mimicking entities, coacervate microdroplets spontaneously form through liquid‐liquid phase separation (LLPS), resulting in condensed and molecularly crowded units without a defined membrane structure [11, 12, 13, 14, 15, 16, 17]. This phenomenon aligns with Oparin's origin‐of‐life hypothesis regarding the coacervation mechanism of prebiotic compartmentalization, which brings together molecules in the prebiotic soup [18]. The majority of coacervate microdroplets are produced via charge‐driven complex coacervation between polyanions and polycations, comprising both synthetic and natural polymers, including proteins, polynucleotides, polysaccharides, and their derivatives [19, 20, 21, 22, 23]. Notably, protein‐derived peptides and peptide derivatives have garnered significant interest from the biomimicry community due to their relatively straightforward chemical structures and functional groups derived from various amino acid side chains, which incorporate multiple moieties responsible for the intermolecular interactions leading to phase separation [24, 25]. Therefore, peptide‐based coacervates can be prepared through either the straightforward self‐coacervation of individual peptides or polymers featuring repeated peptide motifs, or through complex coacervation involving oppositely charged peptides and polyelectrolytes or polymer–peptide hybrids, which have been utilized for microreactors and artificial organelles [26, 27, 28].

Moreover, metal ions derived from primitive inorganic minerals on early Earth likely shaped prebiotic chemistry through peptide‐metal interactions, potentially facilitating the accumulation of metal ions to relatively high concentrations and thereby favoring the emergence and enrichment of a limited set of high‐affinity, extant‐like metallopeptides [29, 30]. Beyond the trajectory of early metallopeptide evolution, recent research has uncovered the role of metal ion‐induced phase separation of proteins into membrane‐less condensates, which are now recognized as potentially central to organizing biochemical pathways or participating in pathogenic processes [31, 32, 33, 34, 35]. For example, calcium ions and zinc ions can either inhibit or promote the LLPS of tau, which is associated with Alzheimer's disease [36, 37]. The underlying mechanism of protein condensation can be attributed to the binding of metal cations with intrinsically disordered N‐terminal domains or histidine residues, which evolved gradually rather than existing during the early stages of life in a prebiotic scenario [38]. Instead, short peptides containing several acidic residues, such as prebiotically plausible aspartic acid and glutamic acid, likely chelated with metal ions to facilitate catalysis [39, 40, 41]. However, the specific role of metal ions in driving the LLPS of simple polypeptides as standalone membrane‐free protocells remains underexplored. In particular, the ways in which coordination modes mediate the kinetic pathways of phase separation into distinct physical compartments in simple peptide‐metal cation systems have not been systematically elucidated.

Herein, we report on a peptide‐based coacervate protocell formed through the LLPS of simple anionic polypeptides, specifically polyaspartic acid, which is induced by electrostatic and coordination interactions with inorganic metal cations. The peptide‐metal ion coacervate microdroplet, using zinc ion as a specific example, exhibits liquid‐like properties that are comparable to those of typical polyelectrolyte coacervates, as evidenced by its droplet fusion behavior and internal molecular fluidity. We demonstrate that the concentration of zinc ions mediates the kinetic pathways of phase separation into gel‐like condensates or droplets, owing to differences in interaction modes confirmed by density functional theory (DFT) calculations and molecular dynamics (MD) simulations. Additionally, the peptide‐metal ion coacervate microdroplet shows effective partitioning capabilities for a diverse range of hydrophobic dyes, hydrophilic biomolecules, and inorganic nanoparticles, which are essential for prebiotic metabolic‐like proto‐systems. Furthermore, the peptide‐zinc ion proto‐system enhances the catalytic efficiency of biomimetic prebiotic peroxidase under light compared to that in bulk solutions, while exhibiting distinct biochemical processes at varying zinc ion concentrations, which indicates that metal ion‐promoted membrane‐free compartments may function as photosensitive carriers capable of capturing light in the near‐visible spectrum within the prebiotic environment. Taken together, our findings suggest that peptide‐metal ion interactions may facilitate the generation of prebiotic compartmentalization through phase separation, advancing the development of primitive bioreactors that involve metals and plausible diverse pathways to prebiotic organization.

2. Results and Discussion

2.1. Metal Cations‐Induced Coacervation of Polypeptide

Considering that aspartic acid, an acidic amino acid, serves as a pivotal metabolite for the synthesis of numerous essential biomolecules and has been hypothesized to have formed under the conditions of primitive Earth, it is proposed as an evolutionary minimal set necessary for the formation of complex peptidic structures [41, 42, 43]. Consequently, negatively charged polyaspartic acid (PASP), with a molecular weight of 10 kDa, was selected as the key building block for membrane‐free microcompartmentalization. Additionally, divalent zinc ion (Zn²⁺) was employed as the oppositely charged component, as Zn²⁺ is not only frequently found in the enzymes of extant biology but also was likely prebiotically available as part of the early Earth niches [29, 44]. Therefore, Zn²⁺ was initially chosen to investigate its role in the formation of coacervates facilitated by peptide‐metal cations interaction. PASP‐Zn²⁺ coacervate microdroplets were generated in a Tris‐HCl buffer containing PASP solution at a final concentration of 5 mg/mL, with Zn²⁺ added at a final concentration of 250 mM (Figure 1a). This process transformed the clear solution into a turbid suspension containing discrete, liquid‐like microdroplets that exhibited well‐defined spherical profiles (Figure 1b,c, and Figure S1). Based on turbidity measurements, the PASP‐Zn²⁺ coacervates could exist in significant quantities within a pH range of 4.0 to 8.0 (Figure S2). However, their stability was limited by the hydrolysis of Zn²⁺, despite the turbidity remaining notably high with further increases in pH. Scanning electron microscopy coupled with energy dispersive spectroscopy (SEM‐EDS) analysis of a single microdroplet, prepared through freeze‐drying, revealed the co‐localization of elements such as carbon (C), oxygen (O), and zinc (Zn) within the coacervate (Figure 1d). In situ observation using time‐lapse microscopy demonstrated that two adjacent droplets at time t₀ coalesced into a single droplet within 4 s, providing evidence of liquid‐like fusion (Figure 1e). Furthermore, fluorescence recovery after photobleaching (FRAP) experiments were conducted by bleaching the signal of FITC‐labeled bovine serum albumin (FITC‐BSA) within the microdroplets to assess internal liquidity. The fluorescence recovery profiles of the bleached area indicated a recovery fraction of nearly 80%, confirming the dynamic rearrangement of internal molecules (Figure 1f).

FIGURE 1.

Zn²⁺‐induced phase separation of PASP into coacervate microdroplets. (a) A schematic representation indicates that PASP undergoes LLPS in the presence of Zn²⁺, driven by the synergistic effects of electrostatic and coordination interactions between Zn²⁺ and the carboxyl groups of PASP. (b, c) Optical observations of the clear PASP solution (b) and confocal fluorescence images confirm the formation of coacervate microdroplets following the addition of Zn²⁺, resulting in a turbid solution (c). (d) SEM‐EDS images of a PASP‐Zn²⁺ coacervate microdroplet reveal the elemental distribution map, indicating the colocalization of C, O, and Zn elements. (e) In situ time‐lapse optical microscopy images capture the fusion process of two adjacent microdroplets (indicated by the red dotted line), which were assessed for fluidity. (f) The FRAP recovery curve of a coacervate microdroplet loaded with FITC‐BSA is presented alongside the corresponding fluorescence microscopy images. (g‐j) Turbidity measurements of the PASP‐Zn²⁺ coacervate microdroplets in the presence of NaCl (g), EDTA (h) 1,6‐HD (i), and urea (j) at varying concentrations. (k) CD spectra of PASP in aqueous solution (red line) and PASP‐Zn²⁺ coacervate microdroplets (blue line). Error bars represent the standard deviations derived from three replicate measurements.

As a typical divalent cation, Zn²⁺ exhibits a positive charge and strong electrophilicity, enabling it to engage in electrostatic and coordination interactions with the carboxyl groups of PASP [45, 46]. These interactions can be evaluated using electrostatic shields and competing chelators, such as sodium chloride (NaCl) and ethylenediaminetetraacetic acid (EDTA). The turbidity of the coacervate solution at final concentrations of 1.6 M for NaCl and 1.9 mM for EDTA was nearly zero, with optical microscopy images demonstrating the disassembly of the coacervate microdroplets (Figure 1g,h and Figures S3 and S4). The re‐addition of Zn²⁺ facilitated the reappearance of the coacervate microdroplets (Figure S3). However, the PASP‐Zn²⁺ coacervates exhibited no significant sensitivity to 1,6‐hexanediol (1,6‐HD) and urea, compounds known to weaken hydrophobic and non‐covalent polar interactions [28], indicating that hydrophobic and hydrogen bonding interactions contributed minimally to the phase separation of PASP (Figure 1i,j). Furthermore, the percentage of β‐sheet in PASP increased slightly from 55.1% to 61.5% after interactions with Zn²⁺, indicating a local conformational rearrangement within a reversible range (Figure 1k). In contrast to classic coacervates that rely on electrostatic interactions as the primary driving force, such as those formed by polydiallyldimethylammonium (PDDA) and adenosine triphosphate (ATP), the stability of PASP‐Zn²⁺ coacervates is superior due to the presence of both coordination and electrostatic interactions (Figures S5 and S6).

Subsequently, various metal ions were tested for their potential to induce LLPS of PASP. These ions included monovalent ions such as Na⁺ and K⁺, divalent ions including Mg²⁺, Ca²⁺, Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, and Cu²⁺, as well as trivalent ions like Cr³⁺ and Fe³⁺. The metal ions were added to the PASP solution at the same final concentration as Zn²⁺, and the resulting coacervation was observed to differ due to the combined effects of the binding affinities between PASP and the metal ions, as viewed under a bright‐field microscope (Figure S7). Notably, Na⁺, K⁺, and Mg²⁺ did not induce LLPS due to the strong electrostatic competition presented by Na⁺ and K⁺, along with the preference of Mg²⁺ to bind with water molecules [47]. In contrast, Cu²⁺, Fe²⁺, and Fe³⁺ were hydrolyzed into precipitates under the same experimental conditions of a pH 7.5 buffer, which approximates physiological conditions, whereas Co²⁺ and Cr³⁺ interacted with PASP to form gel‐like aggregates. The Ni²⁺ solution and PASP solution were miscible at a pH of 7.5; however, they formed coacervates when the pH reached 8.0 (Figure S8). Both Ca²⁺ and Mn²⁺ effectively promoted the coacervation of PASP. However, compared to Ca²⁺, PASP‐Mn²⁺ coacervates exhibited significantly enhanced salt tolerance, suggesting that electrostatic interactions play a dominant role in the formation of PASP‐Ca²⁺ coacervates (Figure S9). Furthermore, another polypeptide rich in acidic residues, specifically polyglutamic acid (pGlu), was investigated for its interaction with Zn²⁺ (Figure S10). The results indicated that pGlu cannot induce LLPS with Zn²⁺, but its presence modifies the phase behavior of PASP.

2.2. Physicochemical Properties of Coacervate Microdroplets Determined by Peptide‐Metal Interactions

Component concentration is a prerequisite for phase separation and directly influences the interactions among the building blocks of coacervates [19, 20, 24]. For instance, when the final concentration of PASP was maintained at 5 mg/mL, both the size of the PASP‐Zn²⁺ coacervate microdroplets and the enrichment of molecules, such as negatively charged RBITC‐labeled GOx (RBITC‐GOx), increased as the concentration of Zn²⁺ rose from 75 to 250 mM (Figures S11 and S12). The internal liquidity of the coacervate microdroplets exhibited no significant differences across varying Zn²⁺ concentrations, with the fluorescence signal recovery consistently exceeding 50% of its initial intensity, indicating similar microenvironments (Figure S13). However, at lower PASP concentrations, the microdroplets tended to become nonspherical and displayed a more amorphous, gel‐like appearance as the concentrations of Zn²⁺ decreased (Figure S14). Based on microscopic images, a phase diagram was constructed, delineating three distinct regions: coacervate, gel‐like condensates, and solution (Figure 2a). With the PASP concentration fixed at 2.5 mg/mL, three Zn²⁺ concentrations within the blue and red regions of the phase diagram—50, 150, and 250 mM—were selected for in situ testing. At a Zn²⁺ concentration of 250 mM, the formation of droplet‐like structures is more likely, while the 50 and 150 mM concentrations correspond more closely to a gel‐like state, as confirmed by optical microscopy images at t₀ in Figure 2b. Data from FRAP experiments illustrate that fluidity was restricted at low Zn²⁺ concentrations (Figure 2c). To compare the differences in the internal environment of coacervate droplets, we employed 8‐anilino‐1‐naphthalenesulfonic acid (ANS), a hydrophobic fluorescent probe that exhibits fluorescence in nonpolar environments. The results indicate that gel‐like condensates formed at lower Zn²⁺ concentration exhibited greater interior hydrophobicity compared to coacervate droplets at higher Zn²⁺ concentration, further suggesting that the strong interactions present at low concentrations lead to increased water release, which accounts for the differences in their sequestration performance of guest molecules (Figures S15 and S16). Furthermore, the phase diagrams of PASP‐metal cation coacervates, particularly those involving Ca²⁺, Mn²⁺, Co²⁺, and Cr³⁺, were drawn in a manner analogous to that of PASP and Zn²⁺ (Figure S17). The observed decrease in critical concentrations for PASP‐Ca²⁺, PASP‐Mn²⁺, PASP‐Co²⁺, and PASP‐Cr³⁺ coacervate microdroplets indicates an enhancement in peptide‐metal interactions, which corresponds with the increase in charge density as the ionic radius decreases in the order of Ca²⁺ > Mn²⁺ > Co²⁺ > Cr³⁺ [48]. However, Co²⁺ and Cr³⁺ formed microdroplets at low concentrations and gel‐like condensates at high concentrations, which significantly deviates from the behavior observed with Zn²⁺, which contradicts the common perception that lower concentrations exert a weaker effect on the interactions among the building blocks of coacervates. Isothermal titration calorimetry (ITC) measurements conducted at low concentrations of Co²⁺ and Zn²⁺ demonstrated a stronger affinity of PASP for Co²⁺ compared to Zn²⁺ (Figure S18). Additionally, the availability of binding sites for Zn²⁺ was found to be lower than that for Co²⁺, suggesting that Zn²⁺ is more likely to bind with multiple PASP chains rather than with a single chain.

FIGURE 2.

Variations in coacervation dependent on Zn²⁺ and PASP concentrations. (a) A phase diagram delineates three distinct regions corresponding to coacervate, gel‐like condensates, and clear solutions, which vary according to the concentrations of PASP and Zn²⁺. (b) In situ time‐lapse optical microscopy images illustrate the formation of coacervate and gel‐like condensates using 2.5 mg/mL PASP and Zn²⁺ concentrations of 50, 150, and 250 mM Zn²⁺, along with their corresponding fusion processes. Scale bars: 10 µm. (c) The FRAP recovery profiles for the coacervate (250 mM Zn²⁺) and gel‐like condensates (50 mM Zn²⁺) at time t₀ are presented in (b). (d) Geometries of the ASP‐Zn²⁺ monocoordination and polycoordination models obtained through DFT calculations. Error bars represent the standard deviations derived from three replicate measurements. (e) Binding energies between PASP and Zn²⁺ in both monocoordination and polycoordination models. (f) Snapshots of the coacervate and gel‐like condensates from MD simulations and the corresponding proportion of coordination quantities. (g) The interaction energies between PASP and Zn²⁺ at concentrations of 50 mM (blue line) and 250 mM (red line).

To investigate the interactions between PASP and Zn²⁺ at two different concentrations, density functional theory (DFT) calculations were initially conducted to elucidate the potential coordination modes between the residue of aspartic acid (Asp) and Zn²⁺. These modes primarily include monocoordination and polycoordination, wherein a Zn²⁺ ion is coordinated with either a single Asp molecule or multiple Asp molecules (Figure 2d). Notably, the polycoordination mode exhibits a higher binding energy (Figure 2e). Subsequently, we conducted molecular dynamics (MD) simulations to explore the molecular details of the assembly regulated by Zn²⁺ concentrations (Figure 2f). Both coordination modes coexist; however, the proportion of coordination quantity differs: monocoordination predominates at high Zn²⁺ concentrations, while polycoordination becomes more prevalent at low Zn²⁺ concentrations. Therefore, in the conformers of PASP/Zn²⁺ at high concentration, Zn²⁺ preferentially coordinates with multiple carbonyl oxygen atoms on a single PASP chain [49], causing the chains to shift from extended to curled conformations, retaining a relatively large number of water molecules and resulting in the formation of microdroplets. Conversely, at low concentrations, Zn²⁺ acts as a bridging center through multi‐dentate coordination, simultaneously binding to carboxyl groups on multiple distinct PASP chains, which facilitates the cross‐linking of originally dispersed molecular chains into a three‐dimensional network, resulting in dense gel‐like condensates. Consistently, as evidenced by the interaction energies in Figure 2g, gel‐like condensates at low Zn²⁺ concentration exhibit stronger interactions between PASP and Zn²⁺ compared to those of coacervates at high Zn²⁺ concentration. Furthermore, we speculate that the presence of water may enhance the thermal motion of the molecular chains, facilitating the transition of the system from a higher‐energy condensate state to a lower‐energy droplet state, thereby aiding in the fusion of coacervate microdroplets. As verified in Figure 2b, the amorphous gel‐like condensates gradually transform into liquid‐like droplets within four minutes, with the duration required for morphological transformation decreasing as the concentration of Zn²⁺ increases.

2.3. Client Recruitment of the Peptide‐Metal Cation Coacervate Microdroplets

The ability to recruit clients is a key characteristic of coacervate microdroplets functioning as membrane‐less protocells, as this recruitment enhances local concentration, thereby facilitating primitive biochemical reactions. To systematically investigate the partitioning properties of peptide‐metal cation coacervates, we selected a diverse range of common clients, including various physicochemical small molecules (both hydrophilic and lipophilic, positively and negatively charged), biological macromolecules (such as polysaccharides, nucleic acids, and proteins), and nanoparticles. Specifically, water‐soluble positively charged rhodamine 6G (R6G) and negatively charged calcein were chosen, along with hydrophobic neutral species such as Nile red. Additionally, larger biomolecules, including dextran, single‐stranded deoxyribonucleic acid (ssDNA), bovine serum albumin (BSA), horseradish peroxidase (HRP), and GOx, as well as nanoparticles like TiO₂ were included. The loading efficiencies were subsequently evaluated by calculating relative partition coefficients (denoted as K _p = F _coacervate/F _background) using confocal imaging, as shown in Figure 3. Five peptide‐metal cation coacervate microdroplets, comprising Zn²⁺, Ca²⁺, Mn²⁺, Co²⁺, and Cr³⁺, exhibited distinct variations in their capacity to sequester the aforementioned molecules. However, all of them displayed suboptimal enrichment performance for R6G, likely due to the excessive positive charges of the metal cation‐based coacervates, while capturing TiO₂ nanoparticles in an aggregated form that may contribute to primitive photocatalysis. Notably, the PASP‐Zn²⁺ coacervate microdroplets exhibited relatively superior enrichment performance for the other molecules compared to the remaining four coacervates. This enhanced performance may be attributed to the combination of strong binding affinity and high internal fluidity, which facilitates both elevated partition coefficients and rapid equilibration (Figures S19 and S20). Consequently, this suggests that Zn²⁺ may be more effective in promoting the transformation of the primitive membrane‐free compartment into a proto‐reactor.

FIGURE 3.

Partitioning of clients within PASP‐metal cation coacervate microdroplets. (a) The confocal laser scanning microscopy images of coacervate microdroplets that formed from PASP at a concentration of 5 mg/mL, in combination with metal ions such as Zn²⁺, Ca²⁺, and Mn²⁺, each at a concentration of 250 mM. In contrast, Co²⁺ and Cr³⁺ were utilized at a lower concentration of 75 mM. Scale bar: 40 µm. (b) The relative partition coefficients of Nile Red, Rhodamine 6G, Calcein, FITC‐Dextran, TAMRA‐labeled ssDNA (TAMRA‐ssDNA), FITC‐labeled BSA (FITC‐BSA), Atto 425 NHS‐labeled HRP (Atto‐HRP), and RBITC‐GOx within the aforementioned coacervates are presented. Error bars represent the standard deviations derived from three replicate measurements.

2.4. Enhanced Light‐Induced Autocatalysis Efficiency in Peptide‐Zinc Ion Coacervates

The Zinc world concept posits that the primordial environment nurturing the earliest life forms was enriched with Zn²⁺ ions, which preferentially associated with ancient RNA and protein molecules, including ribozymes and enzymes that catalyze evolutionarily conserved reactions [50]. This association is believed to have contributed to the elevated levels of Zn observed in modern cells. In this section, we employed G‐Quadruplex/hemin (G4‐hemin) DNAzyme, which functions as a prebiotic peroxidase, and discovered that Amplex Red was unexpectedly converted into resorufin in the presence of Zn²⁺ under 488 nm light (Figure 4a and Figure S21). We speculate that exposure to ultraviolet light triggers the production of reactive oxygen species in the Zn²⁺ solution, as evidenced by the near‐complete inhibition of 3,3′,5,5′‐tetramethylbenzidine (TMB) oxidation upon the addition of Na₂SO₃ to remove dissolved oxygen or EDTA to chelate Zn²⁺, as well as under natural light or sunlight (Figures S22 and S23). Following the compartmentalization formed by the interaction of PASP and Zn²⁺, the catalytic efficiency was enhanced within the coacervate microdroplets, as evidenced by the macroscopically observable red color in the microdroplets compared to the bulk solution (Figure 4b,c). Furthermore, non‐in situ imaging revealed that the red fluorescence emitted from resorufin was gradually generated in both the coacervate microdroplets formed by 250 mM Zn²⁺ with 5 mg/mL and 2.5 mg/mL PASP, as well as in the gel‐like condensates formed by 50 mM Zn²⁺ and 2.5 mg/mL PASP, which were located in the red and blue regions of the phase diagram depicted in Figure 2a, respectively (Figure 4d–f and S24).

FIGURE 4.

Enhanced light‐induced autocatalysis efficiency in peptide‐zinc ion coacervates. (a) The reaction scheme depicting G4‐Hemin DNAzyme facilitates the conversion of Amplex Red into resorufin in the presence of Zn²⁺ and light. (b,c) Photographs show the temporal evolution of the light‐driven reaction system in both the bulk solution (b) and coacervates (c). (d‐f) Non‐in situ time‐lapse fluorescence microscopy images capture the red fluorescence emitted from coacervate microdroplets (d,e) and gel‐like condensates (f). (g) The initial reaction rates for the bulk solution (gray line), gel‐like condensates (blue line), and coacervate microdroplets (pink and red lines) toward the substrate Amplex Red. Error bars represent the standard deviations derived from three replicate measurements. (h) A table summarizes the kinetic parameters of the coacervate microdroplets, gel‐like condensates, and solutions.

To evaluate the kinetic parameters associated with enzymatic activity, kinetic experiments were conducted by varying the concentration of Amplex Red. As shown in Figure 4g, the initial reaction rate exhibited a substrate concentration dependency consistent with the Michaelis‐Menten model. The Michaelis constants (K _m) and the maximum reaction rate (v _max) for the G4‐hemin DNAzyme in coacervate microdroplets, gel‐like condensates, and bulk solution were determined using Lineweaver‐Burk plots (Figures 4h and S25). The elevated K _m values observed in the compartments facilitated by Zn²⁺ suggest that both coacervate microdroplets and gel‐like condensates enhance the enzymes' affinity for substrates compared to that observed in bulk solution. This leads to a significant improvement in v _max, indicating that peptide‐metal cation coacervates function as efficient microreactors for enhancing catalytic efficiency. Moreover, the gel‐like condensates exhibited a higher K _m and a lower v _max compared to those in coacervate microdroplets, further suggesting that proto‐compartments mediate catalysis based on Zn²⁺ concentration. Consequently, we propose that the PASP‐Zn²⁺ coacervate microdroplets may function as photosensitive carriers capable of capturing light in the near‐visible spectrum within the prebiotic environment, thereby potentially facilitating the transfer of energy from the young sun to drive essential proto‐chemical reactions. Additionally, we also tested the light‐induced catalytic performance of other metal ions and found that Mn²⁺ can also play a catalytic role; however, the light‐induced catalysis cannot be confined within PASP‐Mn²⁺ coacervates (Figures S26 and S27).

3. Conclusions

In summary, we have developed a novel membrane‐free protocell model based on coacervates formed from the metal cation‐triggered LLPS of simple anionic polypeptides, particularly exemplified by the PASP‐Zn²⁺ system, which demonstrates liquid‐like properties, client recruitment, and an enhancement in light‐induced catalytic efficiency. Unlike traditional charge‐driven complex coacervation, the physicochemical properties of PASP‐Zn²⁺ coacervates are mediated by the coordination mode determined by zinc ion concentrations, generating microdroplets at high concentrations and gel‐like condensates at low concentrations, which further facilitates distinct light‐induced processes. Overall, our findings present a plausible and diverse pathway for the emergence of prebiotic compartmentalization on early Earth, wherein simple acidic polypeptides and abundant metal cations self‐assemble through peptide‐metal ion interactions. Notably, Asp has been identified in meteorites, and its synthesis under prebiotic conditions has been documented; it also has the potential to evolve into peptides in the presence of minerals [51, 52, 53]. Although the bulk concentration of Zn²⁺ in the Archean ocean was estimated to be below 10 nM, the ‘Zinc World’ hypothesis posits that locally enriched microenvironments, such as hydrothermal ZnS edifices, may have sustained millimolar Zn²⁺ levels, comparable to the intracellular Zn²⁺ content of modern cells [54, 55]. Consequently, the relatively high Zn²⁺ concentrations employed in our study are designed to model such prebiotically plausible, metal‐rich niches, serving as a proof‐of‐concept upper range to ensure robust phase separation and functional exploration. Therefore, the PASP‐Zn²⁺ coacervate proto‐systems not only recapitulate the core properties of modern membraneless organelles but also serve as efficient microreactors for enhancing catalytic reactions, bridging abiotic chemistry and prebiotic organization. Finally, it is anticipated that future research could investigate the evolutionary transition from peptide‐metal coacervates to more complex protocellular systems, such as the integration of genetic materials or the incorporation of prebiotic catalysts, further contributing to elucidating the role of metal ions in the origin and early evolution of life.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: smll73900‐sup‐0001‐SuppMat.docx.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.