Metal ions serve as versatile cofactors that play important roles in supporting catalytic activities of protein and stabilizing the structure of nucleic acid. They are also involved in wide range of biological processes, including enzyme catalysis, hormone secretion, signal transduction, respiration, and photosynthesis [1], [2]. The distinctive properties of metal ions such as various valence states, positive charge, electron-acceptor ability, low-or high-spin configuration, specific ligand affinity, and flexible coordination sphere, contribute to its mobility and diffusivity [1]. Cu2+, Ni2+, Zn2+, Ca2+, Mg2+, Mn2+, Fe2+/3+, Na+, and K+ are the common ions involved in biophysical or biochemical processes, acting as temporary electron carriers for biomolecules [1], [3]. The partake of the metal ions in catalytic activity ultimately contributes to the stabilization of enzyme structural conformation changes, as they could occupy a crucial position in the active site, controlling the redox behaviour of biomolecules [4]. Moreover, the specific chemical properties of each metal cation such as size and coordination dictate the nature and geometry of metal-binding sites. This, in turn, determines the characteristics and chemically neighbouring residues, leading to selectivity and, consequently, greater regulation in function [5].

Apart from the activation of reacting species, the presence of metal ions plays a role in the electronic stabilization of intermediates and transition states. The specific mechanism relies on the chemical nature of both the coordinated species and the catalytic site, whereas the metal ion property that is inevitably exploited is Lewis acidity [6]. It has been reported that almost 30% of folded proteins needs coordination with a metal ion for their physiological function which normally affects the stability and physical properties of the proteins [4], [7]. This is underscored by the Irving-Williams series, which delineates the preferential binding affinity of proteins for divalent metal ions in the order of Mg(II)<Mn(II)<Fe(II)<Co(II)<Ni(II)<Cu(II)>Zn(II), highlighting the variable positioning of copper (Cu2+) and zinc (Zn2+) ions depending on specific biochemical contexts [8]. The critical role of metal ions as cofactors in biomolecular processes has been substantiated through extensive research. For instance, D'Souza et al. reported the influence of Co2+ in activating methionine aminopeptidase (MetAP), an enzyme integral for the selective cleavage of N-terminal methionine from nascent polypeptides, by binding to key residues, thereby facilitating its function as a mononuclear enzyme [9]. Similarly, Guo et al., highlighted the indispensable function of Mg2+ in conferring protection to enzymes within a Cell-Free Protein Synthesis (CFPS) system during lyophilization, attributing to its allosteric protection capabilities that prevent detrimental co-lyophilization effects and enhance substrate binding through induced active enzyme conformations [10]. Further emphasizing the correlation between metal ions and enzymatic activity, Piacham et al. revealed findings on the superior superoxide dismutase (SOD) activity facilitated by Mn2+, surpassing that of Cu2+, Co2+, and Ni2+ in a study involving the bacitracin peptide [11]. This intriguing observation, where metal ions with weaker binding affinities but strong electrostatic charges enhance SOD activity, underscores the complex dynamics governing metalloprotein interactions [12]. The coordination of Mn2+ with specific amino acids—histidine, thiazoline, the N-terminal NH2 group, and glutamic acid—in the metallocyclic peptide highlights the exact structural prerequisites necessary for optimizing enzymatic catalysis and modulating activity.

Identification of metal-binding sites could conventionally be determined via nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, metal-affinity column chromatography, gel electrophoresis, and electrophoretic mobility shift assays, all of which are intricate, require specialized instruments, and are laborious, and time-consuming [13], [14], [15]. The advent of computational approaches has offered alternative methods due to their straightforward, convenient, and rapid analysis of metal-binding site in three-dimensional space. The expanding Protein Data Bank (PDB) intrinsically facilitates the viability of these methods to elucidate the ion selectivity of a protein through its substantial and easily accessible database of protein structures [14]. The identification of metal ion binding sites can generally be categorized into sequence-based and precise structure-based methods such as MIonSite and IonCom, respectively [16].

We recently reported our preliminary findings on the implementation of uricase mimicking mp20 for uric acid determination. The computationally designed uricase from Arthrobacter globiformis (PDB ID.: 2yzb) was exploited to bind specifically to uric acid but was known as an inactive site [17], [18]. Previously, our focus was on the designed 20-residue mini protein (mp20) over other produced peptides (mp40, mp60, mp80, and mp100, each named for their residue counts) was determined by docking studies. These studies demonstrated that mp20 uniquely shares critical residues with native uricase, which are crucial for uric acid substrate interaction. The shared structural feature, particularly in the context of hydrophobic interactions and substrate recognition, underscores the relevance of mp20 in mimicking native uricase activity and provides a rationale for its in-depth study.

Further expanding on our recent research, we have demonstrated the substantial role of copper(II) ions as cofactors in enhancing the electrochemical performance of mp20 [19]. Building upon the methodological achievements of our earlier studies, the current work ventures into exploring the biochemical mechanisms through a colorimetric detection approach, emphasizing the impact of various metal cofactors on the catalytic activity of mp20, particularly copper(II) ions for their biochemical effectiveness.

Through metal ion-binding site prediction and docking studies, we have pinpointed specific interactions between mp20 and copper(II) ions, providing a molecular basis for the observed enhancements in catalytic activity. This transition from electrochemical to colorimetric detection of uric acid illustrates the versatility of the developed bioreceptor across different detection modes, leading to noteworthy results. The introduction of metal cofactors is hypothesized to catalytically activate certain sites on mp20.

A computational analysis on metal ion binding to mp20 was conducted to understand metal ions' involvement and to identify crucial positions at the mini protein's active site. Employing the Metal ion-binding site prediction and docking server (MIB) allowed for the precise identification of metal ion-binding sites and the prediction of metal ion-bound 3D structures through the fragment transformation method, previously discussed by Lin et al [14].. Since the three-dimensional structure and types of residues are often conserved, comparable binding sites could well be identified by comparing the types of residues and their location to those of computationally constructed metal ion-binding residue templates [14], [15].

To meet the prerequisites of an efficient biocatalyst, in line with our previous approach, mp20 was further employed for in-situ encapsulation, one type of immobilization technique utilized within the Zeolitic Imidazolate Framework-8 (ZIF-8). As sub-members of the metal-organic framework (MOF) family, ZIFs, with their zinc ions coordinated by imidazole rings, mimic the structure of zeolites and offer open architectures that act as inert hosts [3]. The properties of ZIFs, including their ordered and adjustable pore structures, minimal toxicity, extensive surface area, and excellent thermal and hydrothermal stability, make them ideal for preserving the structural integrity and catalytic function of biomolecules [3], [20], [21]. The process of encapsulation is influenced by the surface characteristics of biomolecules, while the formation of three-dimensional ZIFs provides a stable microenvironment and resilience to the encapsulated biomolecules [22].

Recent studies have significantly delved into the immobilization of diverse enzyme types onto ZIF-8, including oxidase [23], lipase [24], and peroxidase [25] onto ZIF-8, highlighting two principal immobilization techniques. The first technique involves integrating enzymes into the MOF structure during its formation, known as the de novo or in situ encapsulation approach [26]. This strategy allows for homogeneous distribution and encapsulation of enzymes within the MOF lattice, offering a gentler approach to protect the enzymes from harsh environments, potentially enhancing their stability and activity. The second, post-synthetic technique, introduces enzymes into a synthesized MOF through surface immobilization, covalent linkage, or pore entrapment. These immobilization methods, involving physical or chemical attachment of enzymes onto or within various carriers or supports, effectively address critical limitations associated with the operational stability, recovery, and reuse of enzymes in their native, unbound state.

The significance of enzyme immobilization is underscored by its efficiency in markedly increasing enzymes' resistance to environmental stressors, such as significant pH and temperature variations, thereby prolonging their catalytic lifespan. This attribute enhances the operational efficiency of enzymes and presents a cost-effective alternative by reducing the need for continuous enzyme synthesis. Furthermore, immobilization has been shown to refine enzyme specificity and minimize the risk of product contamination, further enhancing its appeal for various applications. Although advantageous, enzyme immobilization poses challenges. These challenges may manifest as reduced enzyme activity due to potential alterations in the enzyme's natural conformation or steric hindrance, impeding substrate access to the enzyme's active sites.

Examining the encapsulation technique's benefits and potential drawbacks, along with the specific advantages offered by ZIF-8, emphasizes its importance in advancing enzyme technology. Herein, we successfully endeavour to demonstrate the usefulness of the encapsulated mini protein mimicking uricase mp20 by ZIF-8 shells (mp20@ZIF-8), which exhibits prolonged biological functioning in catalyzing uric acid on exposure to perturbation conditions. Fig. 1 summarizes the flow of the process involved in activating of the mp20 and its application for uric acid detection.