# Hydrogen-bonded organic framework biomimetic entrapment allowing non-native biocatalytic activity in enzyme

### Installing the HOF onto Cyt c

For modulating the enzyme’s conformation, we require the artificial HOF to have size-matched cages for enzyme encapsulation, and the nano-biointerface between the enzyme and the cages should be designed. Hydrogen-bonded framework-101 (HOF-101)32,33 is an ideal choice because: 1) its mild assembly condition, and the structural stability originated from its strong layer-by-layer π···π stacking structure, and 2) the periodic carboxylate networks in HOF-101 may favor the formation of nano-biointerface because of the potential hydrogen-bonded interactions between the surface residues of enzymes and the carboxylate networks. Herein, the encapsulation of Cyt c into HOF-101 was realized by a biomimetic mineralization process18,23, wherein the enzyme triggered the HOF-101 nucleation around its surfaces and a net-carboxylate-arranged defective cage was formed for accommodating the enzyme (Supplementary Fig. 1). We envisage that such net-carboxylate-arranged cage may mimic the chaperone cage, because the net-carboxylate arranged in the inner wall of the defective cage results in a highly hydrophilic, net-negatively-charged microenvironment, as like the ones of chaperone cage after protein encapsulation17.

When the HOF-101 was installed onto Cyt c, the apparent color of the material turned from yellow to brownness (Fig. 2a), because of the encapsulation of Cyt c. The standard Bradford assay (Supplementary Fig. 2a) gave a ca. 39 wt% Cyt c content in the nanosystem (Supplementary Table 1). Such a high enzyme loading was also supported by the inductively coupled plasma-mass spectra (ICP-MS) measurement (Supplementary Fig. 2b, c), wherein 0.156 wt% Fe in average was detected in the biocomposites (equaled to 36.2 wt% Cyt c in average). We next designed a surface-adsorption experiment to confirm that Cyt c could not be surface-adsorbed by HOF-101, as evidenced by the UV-Vis and Fourier transform-infrared spectrum (FT-IR) data (details seen in Supplementary Fig. 3). The closer insight into the significant reduction of N2 adsorption amount of Cyt c@HOFs compared to parent HOF-101 (Supplementary Fig. 4) also verified that the Cyt c was indeed encapsulated, rather than surface-adsorbed onto HOF-101. Such HOF-101-encapsulated Cyt c (Cyt c@HOF-101) nanostructure was further supported by other characterizations, including powder X-ray diffraction (Supplementary Fig. 5), thermogravimetric analysis (Supplementary Fig. 6), and small-angle X-ray scattering (Supplementary Fig. 7).

The atomic-level structure of Cyt c@HOF-101 was profiled by low-electron-dose cryoelectron microscopy (cryo-EM, Fig. 2b). The long-range ordered channels with 2.0 nm width were witnessed throughout the biocomposite (Fig. 2b and Supplementary Figs. 8 and 9), which should favor the catalytic substrates diffusion and products transportation. The typical atomic-level information (Fig. 2c), viewed from the [01$${\bar{1}}$$] projection, was clearly presented by the inverse fast Fourier transformation (IFFT) image. The periodically arranged bright spots were assigned to the cavities, which were formed by the intermolecular H-bonded assembly and layer-by-layer π···π stacking of the organic linkers. Such information seen by the cryo-EM was well in agreement with the crystallographic structure of HOF-101, wherein the H-bond connected layers were stacked by A-A stacking, with a ca. 0.37 nm interlamellar spacing (Fig. 2d).

The spatial distribution of Cyt c was profiled by confocal laser scanning microscopy (CLSM) experiments, wherein the Cyt c was labelled by rhodamine b (RhB, a red fluorescence dye). The red fluorescence completely overlaid with the frameworks (Fig. 2e and Supplementary Fig. 10), suggesting the uniformly spatial distribution of Cyt c. These results indicated the exogenous HOF was installed onto Cyt c as we expected (Fig. 2f).

### The conformational structure and functional activity

The interfacial interaction between Cyt c and the installed HOF was then examined. The solid-state nuclear magnetic resonance (ssNMR) spectra of Cyt c@HOF-101 were different from those of the free Cyt c and the physical mixing of Cyt c and HOF-101 (Supplementary Fig. 11). Especially, the chemical shifts of 1H ssNMR ranged from 14 to 15 ppm and 13C ssNMR ranged from 170 to 175 ppm, assigned to the –COOH group of HOF-101, was observed to be shifted in Cyt c@HOF-101. It suggested the additional interaction between HOF-101 and Cyt c in the Cyt c@HOF-101 nanosystem. Furthermore, the closer examination by FT-IR spectra found that, the typical amide I and amide II bands of native Cyt c (ascribed to the C=O stretching and N-H bending34, respectively) were perturbed after encapsulation by HOF-101, further supporting the interfacial interaction between Cyt c and HOF-101 (Supplementary Fig. 12).

The structural change of Cyt c was then examined. We firstly utilized circular dichroism (CD) spectrum to verify that the native secondary structure of Cyt c was well maintained after assembly (Supplementary Fig. 13). The microenvironments of the active center of Cyt c were profiled by UV-visible diffuse reflectance spectroscopy (UV-Vis DRS, Fig. 3a), a sensitive means for heme coordination study. The distinct UV-Vis DRS peaks at 520 nm and 550 nm are the characteristic peaks of hexa-coordinated low-spin heme in native Cyt c35,36, while the peaks at 696 nm is assigned to the characteristic electron transfer peak of heme-M80 residue37,38. Markedly, these three typical absorption bands completely missed in Cyt c@HOF-101, and a typical peak of high spin heme at 625 nm39,40 emerged. It indicated the axially coordinated M80 was removed and a non-native high spin, five-coordinated heme specie was formed. Importantly, the UV-Vis DRS profile of Cyt c@HOF-101 was similar to the native CAT (details were discussed in the molecular dynamics simulations below). Such HOF-101-induced coordination environment change of heme was also verified by ultralow temperature (5 K) electron paramagnetic resonance (EPR) spectroscopy, wherein the high spin ferric hemes41 with g-values of [5.93, 5.66, 1.99] were retained and the low spin ferric hemes42 with g-values of gz (2.93) and gy (2.26) had almost completely disappeared (Fig. 3b). These data suggested the native Cyt c conformation indeed changed after the encapsulation by HOF, and the formed Cyt c@HOF-101 has CAT-like biofunctionality (Fig. 3c), while retained its intrinsic peroxidase activity (Supplementary Fig. 14).

Cyt c@HOF-101 was capable of catalyzing the decomposition of H2O2 into H2O and O2 (experimental details seen in Supplementary Table 2), even though its activity still could not be comparable with the native CAT. This catalytic activities depended on the substrate concentrations (Fig. 3d), and the catalytic kinetics was accorded well with the Michaelis-Menten equation of single substrate enzymatic reaction (Supplementary Fig. 15). We firstly verified the mild liquid phase (deionized water/DMF = 9/1, v/v) used in our synthetic process, could not disturb the native conformation of Cyt c, as evidenced by the intact adsorption bands in UV-Vis spectra of the resultant Cyt c (Supplementary Fig. 16a). In addition, no distinct CAT-like activity was recorded when Cyt c was directly exposed to this condition (Supplementary Fig. 16b). To elucidate the key role of the net-carboxylate-arranged nanocage for the directional modulation of enzyme’s conformation, we also synthesized other prosperous Cyt c-framework nanocomposites through biomimetic mineralization (Fig. 4a), including Cyt c@ZIF-818,43,44,45 (Supplementary Fig. 17) and Cyt c@ZIF-9046 (Supplementary Fig. 18). The UV-Vis DRS profiles suggested that these MOF encapsulation strategies were unable to modulate the conformation of Cyt c (Fig. 4b). Another recently reported Cyt c@NU-1000 nanosystem, wherein the Cyt c was post-infiltrated into the cavity of an intact NU-1000 crystal (Fig. 4a and Supplementary Fig. 19), has shown the possibility of disturbing the heme structure of Cyt c47. However, this mild post-infiltration strategy was also unable to directly modulate the Cyt c heme into a CAT-like species, as seen in the UV-Vis DRS profile (Fig. 4b). Consequently, the formed Cyt c@NU-1000 also displayed very limited CAT-like activity (Fig. 4c). These results together clarified the superiority of our H-boned scaffold, which has net-carboxylate-arranged cages, for the conformational modulation of the enzyme, achieving non-native biofunctionality.

### Insight into the conformation change by computational simulation

Further insight into the conformational structure of Cyt c@HOF-101 was examined by all-atom explicit solvent molecular dynamics (MD) simulations (details seen in Supplementary Discussion and Supplementary Figs. 20–22). The flexible turns with a mass of NH2-rich lysine (such as K87, K86, K72, and K73, region A in Fig. 5a), as well as the short helices with lysine (K8 and K27) and other polar residues of glutamine (Q16), valine (V11) and threonine (T28) of the encapsulated Cyt c (region B in Fig. 5a), were observed to strongly interact with the net-carboxylate arranged in the inner wall of the defective cage via H-bond. The traction by the H-bond between surface residues of Cyt c and HOF-101 resulted in the global conformation change of Cyt c in Cyt c@HOFs, while the conformation of free Cyt c under the same simulating conditions remained unchanged (Supplementary Fig. 23). This H-bonded interface was well in agreement with the aforementioned 1H ssNMR (Supplementary Fig. 11) and FT-IR data (Supplementary Fig. 12).

We then tracked the time-depended microenvironment change of the heme, in terms of the spatial position change of three important amino acids, including the axially coordinated histidine (H18) and methionine (M80) and the distal tyrosine (Y67) (Fig. 5b and Supplementary Figs. 24 and 25). Viewing from region A in Fig. 5a, the dual traction of flexible turn 69–87 of Cyt c by the formed H-bonded interface led to the departure of M80 from the Fe active center of Cyt c (from 2.1 Å to 4.1 Å). The distance of 4.1 Å meant that the axially coordinated M80 turned into the uncoordinated state, which was also supported by the disappeared electron transfer peak of heme-M80 in UV-Vis DRS data (Fig. 3a). Interestingly, we found the distal Y67 was gradually closer to the Fe active center of Cy t c (from 4.8 Å to 2.8 Å, Supplementary Fig. 24) at the same time. When looking into the region B in Fig. 5a, the traction of short helices by the artificial H-bonded interface also caused the spatial departure of H18, and showed the fluctuation of a distance at 2.8–3.3 Å from Fe active center (Supplementary Fig. 24). Distinctly, these conformation changes resulted in a non-native high spin, five-coordinated heme active center, as confirmed in the UV-Vis DRS (Fig. 3a) and EPR spectroscopy (Fig. 3b). We also confirmed that the heme microenvironment of free Cyt c remained unchanged under the same simulating conditions (Supplementary Fig. 25). Importantly, the active center of the nanocage-encapsulated Cyt c was analogous to that of the native CAT, wherein the heme was axially coordinated by tyrosine, with the distal histidine acts as an acid-base catalyst for the O2 generation by the decomposition of H2O248,49.

In the native Cyt c, the heme active center is buried into a flat and narrow cavity (Fig. 5c), and the computational simulation suggested that the H2O2 was hard to access the heme in the native conformation (Supplementary Fig. 26a). Amazingly, in the HOF-101-induced conformation, a larger binding cavity was shaped (Supplementary Fig. 27). This cavity was composed of amino acids arginine 38 (R38), glutamine 42 (Q42), tyrosine 48 (Y48), leucine 35 (L35), leucine 32 (L32), proline 30 (P30), aspartic acid 31 (N31), and histidine 33 (H33), of which the closed R38 amino acid at the top right of the cavity became open. It led to a ca. 6.8 Å pocket that allowed the H2O2 entrance (Fig. 5c and Supplementary Fig. 26b). Importantly, the H2O2 was capable of binding with Fe active site with ca. −4.3 kJ/mol energy (Supplementary Fig. 28a), which is a common stage in both the catalase and peroxidase reactions3,48. In addition, a H-bonded network of H2O2, stabilized by the polar residues within the heme pocket, was seen in Cyt c@HOF-101 (Supplementary Fig. 28b). Such H-bonded network might also facilitate the proton transfer pathway for CAT catalysis49. These simulated data gave a deep insight into the nanocage–modulated conformation change of Cyt c, and might provide a deeper understanding of its CAT-like biological function.

### Catalase-like biocatalysis performance

Among the heme enzymes, Cyt c is the only species wherein the heme macrocycle is covalently linked to the protein skeleton by two disulfide bonds11. Hence, the heme macrocycle of our Cyt c nanosystem is more structurally stable than that in other heme enzymes, such as CAT (Fig. 6a). Besides, the rigid artificial HOF, with ultrahigh chemical stability (Supplementary Fig. 29), can prevent the interior Cyt c from the external stimuli. Meanwhile, the long-range ordered channels of HOF-101 (2.0 nm width) ensure the free diffusion and transportation of many catalytic substrates and their products, yet exclude the interferents such as large macromolecules. Such distinct nanoarchitecture may allow our Cyt c@HOF-101 system to perform catalytic tasks in harsh environments. It is well known that the conformation of native CAT is highly susceptible to external stimulus. When free CAT and Cyt c@HOF-101 were exposed in the scenarios such as non-native pH (Fig. 6b and Supplementary Fig. 30), heating (Fig. 6c and Supplementary Fig. 31), denaturing reagents (urea, hydrolase and heavy metal ions, etc.) and organic solvents (Fig. 6d and Supplementary Fig. 32 and Supplementary Fig. 33) environments for 30 min, respectively, the retained CAT-like bioactivities were examined (experimental details seen in Supplementary Table 3). Herein, the 100% retained activity of Cyt c@HOF-101 or CAT was referred for its original bioactivity in pH = 7 deionized water at room temperature, respectively. And the measurement of bioactivity conversion was based on the change of the initial catalytic rate, which was evaluated by the slope of the kinetic curve (Supplementary Figs. 30–33) in the initial phase from 0 to 40 s. As seen in Fig. 6b–d, Cyt c@HOF-101 retained desirable CAT-like bioactivity. As a comparison, the native CAT was observed to significantly lose its bioactivity under similar treatments. Especially, the native CAT was almost devitalized (the retained bioactivities was less than 10%) when being operated in strong acid conditions (pH = 2), high temperature (80–100 °C) and the environments with ethanol or Cr3+ exposure.

To further elucidate the superiority of our Cyt c@HOF-101 system, we also assembled the CAT@HOF-101 using the similar biomineralization process (Supplementary Fig. 34). The typical amide I and amide II bands of protein appeared in the FT-IR spectra of CAT@HOF-101, suggesting the successful encapsulation of CAT (Supplementary Fig. 34c). In addition, the standard Bradford assay gave a ca. 44 wt% CAT loading in CAT@HOF-101. The typical UV-Vis adsorption bands of CAT heme were well maintained after the encapsulation (Supplementary Fig. 35a), indicating that the native conformation of the heme center was reserved in CAT@HOF-101. However, the activity of CAT was slightly inhibited after encapsulation (Supplementary Fig. 35b), which might be caused by the inevasible inhibition on the substrate diffusion process by the HOF-101 shell. Such activity inhibition of CAT after encapsulation was also observed in CAT@MOFs biocomposites46. As shown in Fig. 6b–d, the stabilities of CAT@HOF-101 were enhanced compared to free CAT, suggesting the protecting effect of the HOF-101 shell in harsh conditions. However, after the same treatments, the activity conversions of Cyt c@HOF-101 significantly outperformed the CAT@HOF-101. These results further suggested that the extraordinary stability of our Cyt c@HOF-101 system partially resulted from the inherently structural robustness of the heme center in Cyt c.

The robust composite construction also made the cold chain-free storage or transportation of enzymes possible, where no obvious CAT-like activity of Cyt c@HOF-101 lost after storage at 35 °C for 5 d (Fig. 6e and Supplementary Fig. 36). Furthermore, the Cyt c@HOF-101 was feasible to be recycled after centrifugation, and retained ca. 70% CAT-like activity after seven times cycles (Supplementary Fig. 37).