Approach of a small protein to the biomimetic bis-(μ-oxo) dicopper active-site installed in MOF-808 pores with restricted access perturbs substrate selectivity of oxidase nanozyme

Advances in nanozymes have taken shape over the past few years in several domains. However, persisting challenging limitations of selectivity, specificity, and efficiency necessitate careful attention to aid in the development of next-generation artificial enzymes. Despite nanozymes having significant therapeutic and biotechnological prospects, the multienzyme mimetic activities can compromise their intended applications. Furthermore, the lack of substrate selectivity can hamper crucial biological pathways. While working on addressing the challenges of nanozymes, in this work, we aim to highlight the interplay between the substrates and bis-(μ-oxo) dicopper active site-installed MOF-808 for selectively mimicking oxidase. This oxidase mimetic with a small pore-aperture (1.4 nm), similar to the opening of enzyme binding pockets, projects a tight control over the dynamics and the reactivity of substrates, making it distinct from the general oxidase nanozymes. Interestingly, the design and the well-regulated activity of this nanozyme effectively thwart DNA from approaching the active site, thereby preventing its oxidative damage. Crucially, we also show that despite these merits, the oxidase selectivity is compromised by small proteins such as cytochrome c (Cyt c), having dimensions larger than the pore aperture of MOF-808. This reaction lucidly produces water molecules as a result of four electron transfer to an oxygen molecule. Such unintended side reactivities warrant special attention as they can perturb redox processes and several cellular energy pathways. Through this study, we provide a close look at designing next-generation artificial enzymes that can address the complex challenges for their utility in advanced applications.


Introduction
Natural enzymes are highly selective, capable of choosing a single substrate amongst the closely related substrates, and are very specic towards a given reaction.2][3] Nevertheless, achieving this level of sophistication in synthetic constructs such as small molecules, supramolecular systems, and nanomaterials remains a formidable challenge.In the frame of catalytic nanomaterials, nanozymes have gained immense interest, mainly due to their potential catalytic features, low cost, and improved stability. 4,5The term 'nanozyme' rst appeared in 2004 for the immobilized triazacyclononane/Zn 2+ on Au nanoparticles used for transphosphorylation activity actually originating from the Zn complex. 6In 2007, the report on magnetic nanoparticles (Fe 3 O 4 ) appeared demonstrating its intrinsic peroxidase-like activity for the rst time. 7][10] However, the eld is still in its infancy due to several critical limitations, including non-biocompatibility, low catalytic activity at physiological pH, and lack of specicity.6][17][18] This catalytic versatility can result in unintended side effects when used in therapeutics, as it can evoke abnormal interactions with several biomolecules, thereby upregulating or downregulating various biological pathways, leading to complications. 19,20The underlying reason could be the well-exposed active site for catalysis, which can readily interact with various substrates, compromising the selectivity of nanozymes.The utilization of molecular imprinting strategy in articial enzyme fabrication has been reported as a unique approach that possesses several advantages in terms of substrate binding affinity. 21,22However, it poses certain limitations such as structural rigidity and poor mass transfer. 23herefore, it is imperative to explore alternative strategies that can overcome these limitations and enable the design of arti-cial enzymes with enhanced performance and specicity.In this regard, understanding the critical details governing the enzymatic properties is essential in achieving catalysts-bydesign, 24,25 rather than general screening of nanomaterials for enzyme-like activities.
Recently, 3D-metal-organic frameworks (MOFs) have emerged as a notable solution, providing a exible approach to designing articial enzymes that incorporate several structural features.These features include nanoconned active sites and a unique diffusion channel environment to impart selectivity and provide a secondary coordination sphere-like microenvironment. 26,27MOFs with interconnected pores closely resemble the substrate channels in enzymes.Recently, MOF-818 was identied for exhibiting specic catechol oxidase-like activity without the peroxidase-like activity. 28Also, Wang et al. 29 have reported an amino acid-treated copper MOF that selectively catalyzes ascorbate, useful for targeted ascorbate sensing in sweat.
Among the various reported MOFs, MOF-808 is deemed to be the most suitable owing to its exceptional structural features and high stability. 30Notably, Yaghi and co-workers reported a remarkable MOF-808 construct with biologically relevant ligands installed in its pore (bis-(m-oxo) dicopper) which has shown catalytic activity for selective methane oxidation. 31urthermore, Zhu and co-workers 32 have explored this construct for catechol oxidase-like activity, which demonstrated recognition and oxidation of chiral catechol substrates by incorporating a chiral histidine ligand.These ndings are highly promising in the domain of designing enzyme mimics with selectivity.
1][52][53][54][55] In this study, we aim to showcase that although the strategies employing pore-engineering of MOFs are essential for regulating selective catalysis of small molecules over complex biomolecules, the activity can still be compromised by small proteins.This could trigger inappropriate activities during the intended function of nanozymes, thus affecting the outcome.Herein, we systematically show that biomimetic bis-(m-oxo) dicopper active sites, closely resembling the dinuclear Cu center from the active site of catechol oxidase and tyrosinase, 56 installed in the pores of MOF-808 (MOF-808-His-Cu) exhibit remarkable substrate-specic oxidase activity towards catechols.As the active site is present inside the adamantane pore with an aperture diameter of 1.4 nm, it serves as an interesting strategy to control the approach of substrates, their dynamics and reactivity.This construct is a selective oxidase mimic that draws a line from other oxidases, peroxidases, catalase and glutathione peroxidase (GPx) nanozymes.8][59][60] Interestingly, in MOF-808-His-Cu, the oxidase-like active sites are under the tight control of pore dimensions (aperture diameter of 1.4 nm) of MOF-808, and therefore, are difficult to access by complex biomolecules like DNA, preventing their ROS-mediated damage.Several oxidase nanozymes, including Cu-containing counterparts, have a potential role in biomedical applications and redox functions. 10,57,59,60However, it is important to note that most of these nanozymes can nonselectively interact with biomolecules, thus they can potentially interfere with various cellular redox processes, electron transfer and energy pathways.Furthermore, such reactivities may lead to a negative impact on the other vital biological processes.Thus, it is highly essential to probe the side reactivities that could impact its clinical translation.In this regard, we thought it worthwhile to check the interactions of a small protein involved in the electron transport chain and energy metabolism, cytochrome c (Cyt c), with MOF-808-His-Cu oxidase nanozyme.While it is understood that the aperture of 1.4 nm helps this construct efficiently keep complex biomolecules away from approaching the active site, we systematically show that proteins such as Cyt c are capable of accessing the active site, compromising substrate selectivity.The approach of Cyt c to MOF-808-His-Cu leads to a redox reaction similar to Cyt c oxidase (COX), majorly leading to four electron oxygen reduction to water.Thus, selectivity in such biomimetic systems is compromised, highlighting a critical look at designing nanozymes with selectivity for clinical applications.While research on imparting selectivity to nanozymes is at the forefront of the domain, a thorough investigation is needed to address the challenges posed by newer solutions.Our research ndings highlight the crucial factors to be considered when designing next-generation enzyme mimetics with high efficiency, substrate and enzyme mimetic selectivity.

Synthesis and characterization of MOF-808-His-Cu
The nanoscale MOF-808-His-Cu was fabricated by following the reported procedure with slight modications with respect to the size of the MOF. 31,61We intended to have a size of the MOF-808 in dimensions below 100 nm because of its relevance in biological applications, especially in cell-based studies. 62The microcrystalline structure of MOF-808 is composed of the secondary basic units (SBUs) Zr 6 O 4 (OH) 4 (COO) 6 (HCOO) 6 connected by a 1,3,5-benzenetricarboxylate (BTC) linker. 63This MOF has two different interconnected pores: the tetrahedral pore (having an internal diameter of 0.48 nm and aperture diameter of 0.12 nm) and the larger adamantane pore (having an internal diameter of 1.8 nm with an aperture diameter of 1.4 nm). 63,64To incorporate L-histidine in the adamantane pores, a post-synthetic modication was performed by binding the carboxylate groups of L-histidine to Zr(IV) in nodes. 65This functionalizes the amino groups to the interior of the pores, serving as the coordination site for Cu metal ions inside the pores (Fig. 1a and S1 †).This ne-tuning and decorating of the pores can serve as reaction pockets analogous to the enzyme binding pockets.As shown by the scanning electron microscopy (SEM) of MOF-808-His-Cu (Fig. 1b), it can be observed that the morphology has been retained aer modication with L- histidine and Cu 2+ ions (Fig. S2a and b †).From the highresolution transmission electron microscopy (HRTEM) image (Fig. 1c, S2c and d †), it can be observed that the particle size of the MOF-808-His-Cu is in the range of 80-100 nm.The energydispersive spectroscopy (EDS) mapping images suggest the uniform distribution of Zr, Cu, O, C, and N elements which further conrms the successful incorporation of L-histidine and Cu 2+ ions in the MOF-808 framework (Fig. 1d).The powder X-ray diffraction (PXRD) analysis revealed that the phase purity and crystallinity of the synthesized MOF-808 were well preserved upon post-functionalization with L-histidine and metalation with Cu 2+ ions (Fig. 1e). 31,63The porosity of the samples was conrmed from the N 2 adsorption-desorption isotherm recorded at 77 K and the Brunauer-Emmett-Teller (BET) surface areas of MOF-808, MOF-808-His, and MOF-808-His-Cu were found to be 868, 752, and 482 m 2 g −1 , respectively (Fig. S3 †).The decrease in the surface area on functionalization can be attributed to the space occupied by the L-histidine and Cu 2+ ions in the pores. 65he successful anchoring of L-histidine and the mode of binding of Cu 2+ ions as Cu-(m-oxo)-Cu species were observed by UV-visible diffuse reectance spectroscopy (UV-vis DRS) (Fig. S4 †). 31,66,67The 1 H NMR spectra of the digested MOF samples further manifest the loading of L-histidine to reveal the degree of functionalization, i.e., 3 histidine ligands per chemical formula as reported earlier (Fig. S5-S7 †). 63,65The loaded Cu content was found to be 7.4 wt% from inductively coupled plasma optical emission spectroscopy (ICPOES).The X-ray photoelectron spectroscopy (XPS) analysis for MOF-808-His-Cu conrms the presence of Zr, C, N, O and, Cu (Fig. S8a †).The deconvoluted spectra of Zr 3d (Fig. S8b †), C 1s (Fig. S8c †), O 1s (Fig. S8d †) and N 1s (Fig. S8e †) and their binding energies are in agreement with previous reports. 68The high-resolution Cu 2p spectrum exhibits the characteristic peaks of Cu 2p 3/2 (932.97 eV) and Cu 2p 1/2 (952.81eV) that are otherwise indicative of Cu(I) and Cu (0) (Fig. S8f †). 69,70][73] Furthermore, the oxidation state of Cu was conrmed by X-ray absorption near-edge structure (XANES). 74The Cu K-edge XANES and extended X-ray absorption ne structure (EXAFS) analysis provide further insights into the oxidation state and coordination environment of Cu.From the Cu K-edge XANES prole (Fig. 1f and S9a †), the absorption edge at about 9006.4 eV is recognized as the dipole-allowed Cu 2+ 1s / 4p transition. 66,75he Fourier transform Cu K-edge EXAFS spectra in the k-range of (3-11) Å −1 and the tting results are presented in Fig. 1g, S9b and c. † The tted parameters are summarized in Table S1, † showing the coordination number of 2.7 assigned to the rst shell of Cu-N/(O) coordination at a distance of 2.00 Å.For the Cu .Cu contribution, the coordination number was xed as 1 and the distance was determined to be 2.52 Å.The results are well in agreement with the literature, 31,66 supporting the presence of bis-(m-oxo) dicopper moieties in the pores of MOF-808.

Catechol oxidase activity and kinetics of MOF-808-His-Cu
To start with, we examined the reported catechol oxidase-like activity for MOF-808-His-Cu, a biomimetic architecture that closely represents the active site of catecholase and tyrosinase enzymes. 76For catechol oxidase-like activity, we employed 3,5di-tert-butylcatechol (DTBC) as the substrate that oxidizes to give 3,5-di-tert-butyl-benzoquinone (DTBQ) (Fig. 2a).As shown in , the peak intensity at 415 nm increases over time, indicating the catechol oxidase activity of MOF-808-His-Cu (12 mg mL −1 ) in phosphate buffer (PB) (pH 7.4) at room temperature (Fig. 2b).In control reactions, MOF-808 and MOF-808-His exhibit no measurable catechol oxidase-like activity, as demonstrated in Fig. 2c.The kinetic study by changing the catalyst concentration (2-12 mg mL −1 ) displayed a proportional enhancement in the activity, suggesting rst-order kinetics (Fig. 2d and S10a †), which was further conrmed by plotting ln rate vs. ln catalyst concentration (Fig. S10b †).As depicted in Fig. 2e and S10c, † the oxidation of DTBC catalyzed by MOF-808-His-Cu resulted in a typical Michaelis-Menten kinetics.The Michaelis-Menten constant (K M ) of MOF-808-His-Cu, obtained from the Lineweaver-Burk plot, is 85.07 mM, while the calculated V max is 39.71 mM min −1 (Fig. S10d †).The low K M value represents an appreciable affinity of MOF-808-His-Cu towards DTBC.The catalytic efficiency (K cat /K M ) of this construct is as high as 550 M −1 s −1 .A comparison of catalytic parameters of reported oxidase nanozymes for the oxidation of catechol substrates is presented in Table S2.† A comparison of these catalytic parameters suggests that MOF-808-His-Cu has an appreciable affinity as indicated above and performs catalysis with relatively higher catalytic efficiency.While the MOF-808-His-Cu shows enzyme-like activity, we tested the stability of the nanozyme by studying the leaching of ions, the effect of pH and temperature, which are crucial for its use in vivo and in vitro under physiological conditions.In the rst experiment, the catalyst was incubated in 50 mM PB (7.4) for 30 minutes.Aer centrifugation, the supernatant was examined for DTBC oxidation activity (Fig. S11a and b †).It was observed that there was no leaching of Cu 2+ from the MOF-808-His-Cu.In the second experiment, we tested the catechol oxidase-like activity of MOF-808-His-Cu treated under different pH conditions (pH 4, 5, 6, 7.4, 8) for 60 min.We observed that the catechol oxidation activity remained unaffected (Fig. S11c †).A slight decrease in activity is observed in the case of MOF-808-His-Cu treated under pH 4, which may be due to the loss of Cu ions at this pH.In the third experiment, we treated MOF-808-His-Cu at different temperatures (25 °C, 60 °C and 80 °C) and then tested its activity.We observed that the MOF-808-His-Cu showed exceptional temperature stability as its enzyme-like activity remained unaffected (Fig. S11d †).However, natural polyphenol oxidases are known to possess very low activity at acidic pH and denature at higher temperatures (the optimum temperature range is 40-50 °C). 77To investigate the catalytic mechanism, we checked the formation of H 2 O 2 in the reaction by a standard 3,3 0 ,5,5 0 -tetramethylbenzidine (TMB)-horseradish peroxidase (HRP) assay.A peak appearing at 650 nm for oxidized TMB conrms the generation of H 2 O 2 as a result of a 2-electron reduction of O 2 (Fig. S11e †).In this context, a catalytic cycle is proposed in Fig. S11f.† Although the oxidation of catechol by natural catechol oxidase results in the corresponding quinone and water as products, 56 MOF-808-His-Cu produces H 2 O 2 in this reaction.

Evaluation of substrate selectivity of MOF-808-His-Cu
0][81][82][83] However, the critical challenge that hinders the development and application of articial enzymes and nanozymes is the lack of substrate selectivity, differing from that of natural enzymes.5][86] To further explore the substrate selectivity of MOF-808-His-Cu, we evaluated its oxidase activity using the above substrates.We initially investigated whether MOF-808-His-Cu catalytically oxidizes glucose.The glucose oxidase (GO) enzyme catalyzes the oxidation of glucose, leading to the production of gluconic acid and H 2 O 2 .The H 2 O 2 generated can be probed at pH 7.4 by HRP, which oxidizes TMB to result in a blue color (Fig. S15a †). 87In an enzyme cascade reaction involving glucose (1 M), 1 U mL −1 GO, HRP, and TMB, we observed that the catalytic oxidation of glucose resulted in an intense blue color (oxidized TMB).However, even aer incubation for 60 min, MOF-808-His-Cu did not exhibit glucose oxidase-like activity (Fig. S15b †).We further tested the consumption of glucose in the absence and presence of MOF-808-His-Cu (for 60 min) using a 3,5-dinitrosalicylic acid (DNS) assay, monitoring absorbance at 540 nm for the formation of 3amino-5-nitrosalicylic acid (Fig. S15c-e †).The amount of glucose was estimated to be the same in control and experimental reactions, conrming no consumption of glucose by MOF-808-His-Cu (Fig. S15c-e †).In another experiment, we checked whether XA can be catalytically oxidized by MOF-808-His-Cu.Xanthine oxidase (XO) catalyzes the oxidation of XA to uric acid (UA) and produces H 2 O 2 (Fig. S16a †).This oxidation process can be assessed by probing the produced H 2 O 2 employing HRP-coupled reactions and analyzing uorescence read-out.One such assay is 10-acetyl-3,7-dihydroxyphenoxazine (amplex red) (AR)/HRP, which effectively probes H 2 O 2 formation, resulting in uorescent resorun with emission at 583 nm. 88A positive control was employed in the presence of XO (33.6 mU mL −1 ) under similar reaction conditions, which resulted in an intense pink color.Conversely, no emission was observed at 583 nm for XO assay when MOF-808-His-Cu was present (Fig. S16b †).We further tested the consumption of XA in the presence of MOF-808-His-Cu (for 60 min) by directly monitoring its absorbance at 270 nm in the UV-vis spectra (Fig. S16c and d †).We observed no change in the absorbance at 270 nm, conrming no consumption of XA by MOF-808-His-Cu (Fig. S16c and d †).Urate oxidase is another copper-containing enzyme that possesses a type-2 copper binding site. 89In this study, to determine the oxidation of UA, we monitored the consumption of UA in the presence of MOF-808-His-Cu at pH 7.4 (Fig. S17a †).There was no change in the absorbance of UA peaks on incubation with MOF-808-His-Cu for 60 min, indicating no signicant UA oxidation (Fig. S17b and c †). 90 NADH and NADPH oxidases are another class of oxidases that are involved in the oxidation of coenzymes.Considering the vital role of NADH/NAD + and NADPH/NADP + as redox couples in maintaining the cellular redox potential in the signalling pathway, mitochondrial function, and numerous other biological functions, 91 we thought it worthwhile to evaluate if MOF-808-His-Cu can catalytically oxidize NADH (Fig. S18a †) and NADPH (Fig. S19a †) by UV-vis spectroscopy.Our results indicate that there was no change in the absorbance in the UVvis spectra of both NADH (Fig. S18b and c †) and NADPH (Fig. S19b and c †), indicating no signicant oxidation in the presence of MOF-808-His-Cu.
Substrates such as L-tyrosine are oxidatively converted to dityrosine by dual oxidases (DUOX). 92Therefore, we introduced L-tyrosine in our reaction and assessed the formation of dityrosine in the presence of MOF-808-His-Cu (Fig. S20a †).However, no catalytic activity was noted, as we did not observe the peak at 410 nm accounting for dityrosine in the uorescence spectra (Fig. S20b †).
In a practical application scenario, multiple substrates exist simultaneously in the biological environment.Therefore, we evaluated the catechol oxidation by MOF-808-His-Cu in the presence of multiple substrates including DTBC, glucose, XA, Ty, and, UA.We observed a remarkable selectivity in the preferential oxidation of DTBC by MOF-808-His-Cu (Fig. S21a-d †).However, the rate of oxidation was slightly decreased in comparison to DTBC oxidation catalyzed by MOF-808-His-Cu as multiple types of substrates were present simultaneously.
Among all the substrates tested for oxidase activity, only catechol-based substrates were oxidized in the presence of MOF-808-His-Cu at pH 7.4 (Fig. 2f).This suggests that the biomimetic bis-(m-oxo) dicopper with L-histidine, which closely resembles the Type-3 copper 93 center found in catecholase, has some inuential role in selectively oxidizing catechol-based substrates.

Evaluation of the enzyme mimicking selectivity of MOF-808-His-Cu
While enzyme mimetic activities that are complementary to each other are benecial, antagonistic activities can compromise the intended applications.94,95 The nanozymes that are composed of copper-based systems have also been shown to exhibit peroxidase, superoxide dismutase (SOD), catalase, and GPx-like activities together. 96,97In another study, ultra-small Cu 5.4 O nanoparticles displayed SOD-, catalase-and GPx-like activity. 13More recently, Geng and co-workers have described a copper-based single-atom nanozyme exhibiting superior multi-enzymatic properties, including peroxidase, oxidase, and catalase-like activity. 14However, we thought it worthwhile to evaluate the enzyme-mimicking selectivity of MOF-808-His-Cu.
][100] AR was utilized as a substrate to assess the oxidase and peroxidase-like activity under physiological conditions (pH 7.4). 88Our ndings, as demonstrated in Fig. S22a, b, S23a and b, † indicate that MOF-808-His-Cu did not exhibit oxidase and peroxidase-like activity, respectively, for the AR substrate at pH 7.4.In another control reaction, HRP showed immediate oxidation of AR to resorun in the presence of H 2 O 2 as monitored by uorescence spectroscopy.We further evaluated the catalase-like activity by monitoring the decrease in the absorbance of H 2 O 2 at 240 nm using kinetics mode in UVvis spectroscopy (Fig. S24a †).Our observation reveals that there is no change in the H 2 O 2 absorbance in the presence of 12 mg mL −1 of MOF-808-His-Cu.This suggests that MOF-808-His-Cu does not display catalase-like activity (Fig. S24b †).GPx is acknowledged as another antioxidant enzyme crucial for regulating H 2 O 2 levels by utilizing the endogenous thiol, glutathione (GSH). 101It catalyzes the reduction of H 2 O 2 to H 2 O, in turn converting GSH to glutathione disulde (GSSG).Consequently, GSSG is reduced to GSH by glutathione reductase (GR) using coenzyme NADPH (Fig. S25a †).We conducted the GR-based assay by following the decrease in NADPH concentration at 340 nm.However, MOF-808-His-Cu showed no decrease in NADPH concentration, thus revealing no GPx-like catalytic activity of the nanozyme (Fig. S25b †).Another vital antioxidant enzyme-like activity is the superoxide dismutase (SOD) activity, 102,103 which involves the dismutation of superoxide anion radical (O À$ 2 ) to O 2 and H 2 O 2 .The SOD activity was investigated by probing the O À$ 2 using water-soluble WST-1 dye (2-(4-iodophenyl)-3-(4-nitrophenyl)-5(2,4-disulfophenyl)-2Htetrazolium) (Fig. S26a †).The O À$ 2 generated from the reaction of XA and XO reduces WST-1 to formazan (yellow color), which displays a characteristic absorption peak at 440 nm. 98However, there was no formazan production observed for the reaction between XA and XO in the presence of MOF-808-His-Cu, indicating that this construct exhibits SOD-like activity (Fig. S26b c †).

Evaluation of DNA damage by side reactions of MOF-808-His-Cu
Amongst the various ROS, hydroxyl radicals are highly reactive, inducing damage to biomacromolecules (such as DNA, proteins, and lipids)., along with other controls (Fig. 2gh).The reaction mixtures were incubated for 2 h at 25 °C and assessed by agarose gel electrophoresis.In the DNA cleavage studies performed without H 2 O 2 , we observed no change in band intensity (Fig. S27c †).While CuSO 4 as a control degraded the DNA completely under our experimental conditions, it is interesting to note that the DNA remained completely intact even in the presence of MOF-808-His-Cu and H 2 O 2 (lane 4) (Fig. 2i).These results are in contrast to the Fenton reaction that was probed using TPA, as discussed above.Aer careful analysis of the results of these experiments, we came to the conclusion that the DNA being a large molecule cannot enter the pores of MOF-808.This also further conrms that the hydroxyl radicals generated at the active site of MOF-808-His-Cu, as a result of the Fenton reaction, are prevented from cage-escape into the reaction environment.In the case of TPA, being a signicantly smaller molecule than the size of the pore aperture of MOF-808, it easily enters the pores and reacts with hydroxyl radicals, forming hydroxy TPA.From these experiments, we infer that the MOF-808-His-Cu projects a tight control over substrate selectivity based on the size of molecules.This prompted us to investigate the fate of small proteins in the presence of this nanozyme.
Probing interactions of small protein Cyt c with MOF-808-His-Cu Considering the small pore-aperture of MOF-808, we hypothesized that the accessibility and hence, the reactivity of smaller proteins would be impossible.To test this, we selected Cyt c which is a smaller heme protein with the dimension of 2.6 nm × 3.2 nm × 3.3 nm (Fig. 3b). 106Cyt c is a mobile single electron carrier that is present in the intermembrane mitochondrial spaces.It plays a key role in shuttling electrons between complex III and cytochrome c oxidase (COX) in complex IV.This process of electron transfer in the electron transport chain is accompanied by the continuous pumping of protons across the membrane, which is vital for mitochondrial ATP production (Fig. 3a). 107In a study by Chen et al. 108 they demonstrated how Cyt c protein enters the MOF cavities with pore sizes less than the dimensions of Cyt c.Recent studies by Triphathi et al. have shown the electrical unfolding of Cyt c through silicon nitride nanopores having diameters of 1.5 to 5.5 nm. 109Their study found that Cyt c could squeeze through the nanopore of 2.5 nm diameter, while fully unfolded protein threading occurred at the mouth of the nanopore with diameters 1.5 and 2.0 nm.In our study, to test the accessibility of Cyt c through the restricted pore, MOF-808-His-Cu could be the best choice as it also has the active center containing copper-histidine moieties, similar to the COX enzyme containing the copper redox centers with an imidazole-based ligand system. 110he COX activity was studied using reduced Cyt c, which was generated by reducing the oxidized Cyt c with sodium dithionite in 50 mM PB (pH 7.4) and further puried using a Sephadex G-25 desalting column.The activity is monitored using UV-vis spectroscopy, following a decrease in the Soret band at 409 nm and a-band at 550 nm, which are the characteristic peaks assigned for the oxidation of reduced Cyt c. 111 In our study, reduced Cyt c (10 mM) in the presence of MOF-808-His-Cu (12 mg mL −1 ) showed an effective decrease in the a-band (550 nm) intensity and a blue shi of Soret band (at 409 nm) under physiologically relevant conditions (50 mM PB, pH 7.4).The decrease in intensity at 550 nm was consistent with the oxidation of Cyt c (Fig. 3c and S28a †), which resulted in a color change from pink to yellowish-orange (Fig. S28b †).However, minimal auto-oxidation of Cyt c was evident in the absence of MOF-808-His-Cu even aer 60 min (Fig. S28c †).Also, MOF-808 (without Cu active sites) did not possess any COX activity under identical reaction conditions (Fig. S28d †).To validate that the COX activity displayed by MOF-808-His-Cu was not due to the leaching of Cu 2+ ions, the catalyst was incubated in PB, followed by centrifugation.The supernatant did not display any COX activity, as evident from Fig. S29a.† In comparison to MOF-808-His-Cu, the equivalent amounts of free Cu 2+ ions exhibited remarkably higher COX activity (Fig. 3d).Thus, it was conrmed that the COX activity was not due to the leaching of Cu 2+ ions.Notably, when the COX activity was carried out in the N 2 -saturated buffer, an appreciable decrease in the activity was noted, revealing the role of O 2 in the reaction (Fig. 3e).
Interestingly, with the increasing concentration of nanozyme (1-17 mg mL −1 ), while keeping the Cyt c concentration xed as 10 mM, we observed that the rate improved with an increase in the concentration of the catalyst, but was not equally proportional (Fig. 4a, S30a and b †).In another case, when we increased the concentration of Cyt c (2.5-50 mM) in the presence of 12 mg mL −1 of catalyst (Fig. 4b and S30c †), we observed a non-typical sigmoidal prole of the curve, distinguishing from the Michaelis-Menten kinetics observed for the natural COX. 112,113nitially, the activity is very low for ∼2.5-15 mM, while the catalytic efficiency gradually increases aer 20 mM and proceeds further toward saturation with an increase in the concentration of Cyt c to about 50 mM (Fig. 4b).This is in contrast to our study on utilizing small molecule DTBC as a substrate which follows rst-order kinetics with increasing catalyst concentration and a Michaelis-Menten curve upon increasing DTBC concentration (Fig. 2e).Therefore, this suggests that the oxidation of Cyt c follows a distinct mechanism in its interaction with the active site located within the pore.The interaction of Cyt c with the catalyst was conrmed by circular dichroism (CD) studies.In general, in the wavelength range of 200-300 nm, the a-helical composition of the protein is probed, while the wavelength range of 300-500 nm demonstrates the changes near the heme environment. 114There is a signicant difference in the absorption features of reduced and oxidized Cyt c in the 300-500 nm region (Fig. 4c).During Cyt c oxidation in the presence of MOF-808-His-Cu (aer 15 min incubation), no changes in the feature of the CD spectrum of reduced Cyt c were observed in comparison to the reduced Cyt c alone (control) in the 200-300 nm region (Fig. S31a and b †).Nevertheless, a signicant change was observed in the region 300-500 nm, which directly reects the changes in the heme environment and is consistent with the oxidation of reduced Cyt c (Fig. S31c and d †).Collectively, these results suggest that during the catalytic oxidation, the structure of Cyt c is not altered by MOF-808-His-Cu.This is also evident from the nature of CD spectra of oxidized Cyt c in the presence of MOF-808-His-Cu (Fig. S31a and c †).
To further elucidate the impact of catalyst size on the approach of the Cyt c protein, we evaluated the COX activity for ∼500 nm scale MOF-808-His-Cu (Fig. S32a †).Our ndings, as shown in Fig. S32b and c, † indicate that the activity and rate were comparable to that observed in the nanoscale MOF-808-His-Cu.These observations suggest that irrespective of MOF-808 size, the approach of Cyt c remains consistent because of the identical pore size and aperture.In mitochondria, the oxidized Cyt c molecules receive electrons from complex III of the electron transport chain, which further transfers electrons to COX.This follows the reduction of O 2 to H 2 O via a four-electron pathway at the active site of COX. 107owever, mitochondria also contribute to cellular oxidative stress by producing ROS such as O À$ 2 with complex I and III being the major sites (Fig. 3a). 115SOD then converts the generated O À$ 2 to peroxide, which is further converted to water and O 2 by catalase.Therefore, the antioxidant system has a tight control over ROS detoxication. 116Thus, it is vital to assess the generation of ROS such as O À$ 2 or hydroxyl radical, during Cyt c oxidation.To understand the plausible mechanism for Cyt c oxidation in the presence of MOF-808-His-Cu, we rst investigated if the addition of SOD in the reaction assay has any effect on the rate of Cyt c oxidation (Fig. S33a †).Under this condition, we did not observe any noticeable change in the activity, and hence, we infer that the O À$ 2 are not generated during the reaction.Furthermore, the SOD-like activity of MOF-808-His-Cu, as discussed earlier, is complementary to Cyt c oxidation.Next, we evaluated the formation of H 2 O 2 during the Cyt c oxidation by the AR/HRP assay (Fig. S33b †). 117Through the AR/HRP assay, we noticed that there was marginal peroxide formation (0.58 mM) as a result of partial reduction of O 2 , following a twoelectron pathway (Fig. 4d and S33c †).This was also supported by the experiment carried out using exogenous addition of catalase during the reaction that resulted in no change in the MOF-808-His-Cu catalyzed Cyt c oxidation activity (Fig. S33a †).Finally, we tested if hydroxyl radicals are generated from the marginally produced H 2 O 2 .The formation of hydroxyl radicals was probed by TPA.No apparent emission was observed in the catalytic system for MOF-808-His-Cu + Cyt c + TPA.When Cyt c oxidation was performed with an equivalent amount of Cu 2+ in the presence of TPA, an intense uorescence emission at 425 nm was observed (Fig. S33d †).Thus, from this experiment, it can be concluded that no hydroxyl radical species are generated during the oxidation of Cyt c in the presence of MOF-808-His-Cu.Through a series of experiments and characterization of MOF-808-His-Cu, we have elucidated a plausible catalytic mechanism (Fig. 4e).Overall, in a major pathway, the oxidation of Cyt c by MOF-808-His-Cu results in the reduction of O 2 to water by the transfer of four-electrons from four Cyt c molecules.The minor pathway, i.e., production of H 2 O 2 results from the two-electron reduction of O 2 molecule as shown in Fig. 4e.However, this is in contrast to the catechol oxidase-like activity of MOF-808-His-Cu using DTBC as a substrate that results in the generation of H 2 O 2 .This could be due to the difference in electron transfer mechanisms that depend on the type of substrates involved in the activity.This work mainly focuses on studies pertaining to the selectivity of nanozymes and evaluating critical factors that can compromise applications.Therefore, the interaction of small proteins with porous structure-based nanozymes requires careful attention.The detailed investigation of the mechanism by which Cyt c accesses the active site of MOF-808-His-Cu will be discussed in the follow-up work.

Conclusions
The growing interest in nanozymes highlights their potential in various applications.However, limitations associated with their specicity and selectivity require careful attention.Despite considerable efforts to develop articial enzymes to achieve specicity comparable to natural enzymes, it remains a longstanding challenge, necessitating further advancement in this area.MOFs have gained signicant attention as articial enzyme mimics since they offer tuning of the structural features within a single construct.The 3D structure with nanoconnement and exactly uniform repeating pores, along with the substrate channels, can play an inuential role in enhancing the catalytic performance.Furthermore, modulating the catalytic microenvironment with ligands similar to natural enzyme counterparts, and substrate recognition sites can help improve the structure-activity relationship of the nanozyme.The multienzymatic behavior can, in turn, result in a decrease in catalytic efficiency, thus achieving substrate selectivity, and singleenzyme mimic features can play a vital role in improving the catalytic efficiency with high specicity.Furthermore, as nanozymes offer promising therapeutic and biotechnological prospects, the antagonistic multienzyme mimetic activities and lack of substrate selectivity can negatively affect their intended applications.Through our investigation, we have shown the signicance of pore-engineering strategies of MOFs in achieving selectivity in enzyme mimics.The biomimetic active site (bis-(m-oxo) dicopper) within the pore of MOF-808 exhibited an impressive substrate-selective oxidase activity by predominantly oxidizing the catechol-like substrates.This substrate selective oxidase mimic, wherein the approach and the reactivity of the substrates are tightly controlled by the small poreopening (1.4 nm) as inspired by the opening of enzyme binding pockets, makes it distinct from the other oxidase mimic in achieving substrate selectivity.Despite this well-regulated access to the active sites and effective prevention of approach of DNA to the active site and its ROS-mediated damage, the small proteins such as Cyt c having dimensions larger than the pore-opening of MOF-808 can access the active site and compromise the selectivity of the nanozyme.As it is important to acknowledge this limitation of MOF-808-His-Cu, our studies on the detailed mechanism of interaction and the reactivity of this exible protein (Cyt c) will be discussed in the follow-up work.The unusual approach of redox protein towards poreengineered nanozymes could hamper several redox and energy metabolism pathways and inuence unintended reactions, thus, it warrants special attention.This study signicantly points to setting guidelines for the development of nextgeneration enzyme mimetics with improved strategies, thereby advancing their potential use in several biological applications.

Fig. 1
Fig. 1 (a) Schematic illustration of the synthesis of MOF-808-His-Cu with the biomimetic bis-(m-oxo) dicopper active site installed inside the adamantane pore and its expanded view.(b) SEM and (c) TEM images of MOF-808-His-Cu.(d) Elemental mapping highlighting the spatial distribution of Zr (green), O (pink), C (yellow), N (blue), and Cu (red) in MOF-808-His-Cu.(e) PXRD spectra of synthesized materials compared with the simulated MOF-808 crystal structure.(f) Normalized Cu K-edge XANES spectra of MOF-808-His-Cu, along with the reference compounds (Cu metal and CuO).(g) k 2 -weighted Cu-EXAFS c(R) spectra of MOF-808-His-Cu, along with the reference compounds.

Fig. 2
Fig. 2 (a) Illustration depicting the catecholase-like activity of MOF-808-His-Cu.(b) Time-drive mode UV-vis absorption spectra of DTBC (200 mM) catalyzed by MOF-808-His-Cu (12 mg mL −1 ).(c) Bar diagram showing the initial rates of DTBC oxidation under different assay conditions.(d) The effect of MOF-808-His-Cu concentration on DTBC oxidation displays first-order kinetics.(e) Michaelis-Menten plot by varying DTBC concentration (30-200 mM) at a constant concentration of MOF-808-His-Cu (12 mg mL −1 ).(f) Illustrative representation showing oxidase substrate selectivity and enzyme mimicking selectivity exhibited by MOF-808-His-Cu.(g) DNA cleavage pattern of the agarose gel electrophoresis, in the presence of H 2 O 2 (1 mM), in PB solution (pH 7.4).(h) A diagram depicting the DNA cleavage in the presence of free Cu 2+ ions, while DNA remaining intact in the presence of MOF-808-His-Cu and H 2 O 2 (1 mM).(i) The extent of % DNA intact plot of the corresponding lanes.

Fig. 3
Fig. 3 (a) Schematic diagram showing the electron transport chain in mitochondria, representing various complexes involved in the systematic transfer of electrons to Cyt c oxidase (complex IV), where O 2 is reduced to H 2 O via a four-electron pathway.(b) Diagram depicting that Cyt c can access the active site of MOF-808-His-Cu despite the large dimensions relative to MOF-808 pore opening.(c) UV-vis absorption spectra of ferrous Cyt c (10 mM) in the presence of MOF-808-His-Cu (12 mg mL −1 ) at pH 7.4.(d) Time-dependent absorbance spectra of Cyt c oxidation in the presence of MOF-808-His-Cu and Cu 2+ ions monitored at 550 nm.(e) Initial rates of Cyt c (10 mM) oxidation in the presence of MOF-808-His-Cu (12 mg mL −1 ) under air-and N 2 -saturated reaction conditions.

Fig. 4
Fig. 4 (a) Effect of varying concentration of MOF-808-His-Cu on Cyt c (10 mM) oxidation at pH 7.4.(b) The sigmoidal curve was obtained for the varying concentrations of Cyt c (2.5-50 mM) in the presence of a fixed amount of MOF-808-His-Cu (12 mg mL −1 ).(c) The oxidation of Cyt c (Fe 2+ ) to Cyt c (Fe 3+ ) in the presence of MOF-808-His-Cu was studied by circular dichroism (CD).(d) The AR/HRP assay for the detection of H 2 O 2 formation during Cyt c oxidation in the presence of MOF-808-His-Cu.(e) The possible side reactions, ROS produced during Cyt c oxidation and the plausible catalytic cycle for the Cyt c oxidation catalyzed by MOF-808-His-Cu.
104,105Transition metal ions like Fe 3+ and Cu 2+ exhibit the Fenton reaction in the presence of H 2 O 2 ,