Behind the Paper

Spatial engineering of single-atom Fe adjacent to Cu-assisted nanozymes for biomimetic O2 activation

Ying Wang Mar 22, 2024

Single-atom nanozymes (SAzymes), characterized by well-defined configuration and maximum atom-utilization efficiency, hold substantial promise in bridging the gap between heterogenous nanomaterials and homogenous enzymes, opening a new era of next-generation nanozymes. During my Ph.D. studies under the supervision of Prof. Xiaoqiang Cui at the Jilin University, I developed Fe oxidase-like SAzymes with well-screened Fe–N3 units by systematically investigating the structure-dependent enzymatic activity1,2. To address the limitations of poor specificity inherent in SAzymes, I embarked on a quest to elucidate the relationship between peroxidase-like specificity and coordination number configurations of Mo SAzymes (Fig. 1a)3. Following the completion of my Ph.D., under the mentorship of Prof. Jeong Woo Han at Pohang University of Science and Technology, I endeavored to modulate the microenvironment of SAzymes, striving to control their multi-enzyme properties. This endeavor culminated in the fabrication of Mn SAzymes, showcasing impressive oxidase, peroxidase, and glutathione oxidase-like activities (Fig. 1b)4.

Figure 1 Schematic diagram of coordination number regulation for (a) the peroxidase-like specificity of Mo SAzymes and (b) the multi-enzymatic activities of Mn SAzymes3, 4.

Previous research efforts have focused on enhancing the intrinsic enzyme-like activities and specificity of SAzymes at the atomic level. The development of SAzymes with dual sites remains in its infancy, necessitating further progress to bridge the gap between heterogeneous and homogeneous biocatalysis. Drawing inspiration from biological enzyme systems and under the guidance of Prof. Kwok-Yin Wong at the Hong Kong Polytechnic University, I delved into the intricacies of natural cytochrome c oxidases (CcO). These terminal metalloenzymes, comprising hemes (heme a and heme a3) and copper centers (CuA and CuB), epitomize homogeneous enzymatic features through the synergistic integration of well-arranged components within a three-dimensional (3D) pocket, which includes central metal units, covalently bonded ligands in the first coordination shell, and appropriate binding sites through non-covalent interactions in the second shell coordination. It is noteworthy that introducing axial ligand coordination as cofactors into single-atom configuration has shown promising results for engineering enzyme-like performances. However, SAzymes designed using reported spatial regulation strategies have primarily focused on mimicking partial bio-inspired structures of natural enzymes, largely overlooking the multi-spatial dimensionality and homogeneous enzymatic pathway. In essence, it is both feasible and meaningful to explore a more versatile spatial engineering strategy that can concurrently integrate heterogeneous SAzyme configurations and homogeneous enzyme-like mechanisms, thereby unlocking their full O2 activation capacity.

In this work, we developed a comprehensive spatial engineering strategy to fabricate dual-site SAzymes (Fig. 2a). These SAzymes incorporate single atom Fe active centers (Fe–N4) and Cu atomic species (Cu–N4) with distinct spatial configurations. Both experiments and theoretical results indicate the dual-site SAzyme featuring vertically stacked Fe–N4 and Cu–N4 geometry (FePc@2D-Cu–N–C) exhibits stronger electronic coupling and synergistic interaction compared with planar Fe–Cu pairs in 2D architectures (2D-FeCu–N–C), thus enabling a similar electron transfer process to that observed in natural CcO. This spatial configuration of FePc@2D-Cu-N-C SAzyme leads to enhanced oxidase-like performance, surpassing the conventional 2D-FeCu-N-C and single-atomic Fe/Cu counterparts. Systematic kinetic investigations unveiled a highly favorable binding affinity to enzyme substrates and strong O2 activation on the FePc@2D-Cu–N–C. Similar to the natural CcO-like reaction pathway, the FePc@2D-Cu–N–C SAzyme facilitates four-electron O2 reduction to H2O, achieving homogenous biomimetic O2 activation (Fig. 2b). In a proof-of-concept application of drug metabolism, this as-developed FePc@2D-Cu–N–C SAzyme also demonstrates remarkable cytochrome P450 3A4 (CYP3A4)-like activities (Fig. 2c). As a potential alternative to CYP3A4, the proposed FePc@2D-Cu–N–C SAzyme can be employed to regulate the drug–drug interaction (DDI) and explore the corresponding mechanism of inhibitory behaviors.

Figure 2 Schematic diagram of (a) spatial engineering strategy, (b) the O2 activation process of natural CcO and FePc@2D-Cu–N–C SAzymes, and (c) the dehydrogenation of 1,4-dihydropyridine (1,4-DHP) catalyzed by CYP3A4 natural enzyme and FePc@2D-Cu–N–C.

This work cannot be done without the valuable input and close collaboration from Prof. Kwok-Yin Wong (The Hong Kong Polytechnic University), Dr. Lawrence Yoon Suk Lee (The Hong Kong Polytechnic University), Dr. Songhua Cai (The Hong Kong Polytechnic University), Prof. Jingxiang Zhao (Harbin Normal University), Dr. Kug-Seung Lee (Pohang Accelerator Laboratory), Dr. Vinod K. Paidi (European Synchrotron Radiation Facility), Prof. Xiaoqiang Cui (Jilin University), Dr. Guangri Jia (Jilin University), Dr. Weizhen Wang (The Hong Kong Polytechnic University), Dr. Yong Wang (The Hong Kong Polytechnic University), and Tingyu Yan (Harbin Normal University). I also would like to sincerely appreciate the critical suggestions of all the reviewers, which greatly improve the quality of the work.

To learn more about our work, please follow the paper “Spatial engineering of single-atom Fe adjacent to Cu-assisted nanozymes for biomimetic O2 activation” published in Nature Communications.

References:

  1. Wang Y, Du R, Lee LYS, Wong K-Y. Rational design and structural engineering of heterogeneous single-atom nanozyme for biosensing. Biosens. Bioelectron. 216, 114662 (2022).
  2. Wang Y, Zhang Z, Jia G, Zheng L, Zhao J, Cui X. Elucidating the mechanism of the structure-dependent enzymatic activity of Fe–N/C oxidase mimics. Chem. Commun. 55, 5271-5274 (2019).
  3. Wang Y, et al. Coordination number regulation of molybdenum single-atom nanozyme peroxidase-like specificity. Chem 7, 436-449 (2021).
  4. Wang Y, et al. Tuning local coordination environments of manganese single-atom nanozymes with multi-enzyme properties for selective colorimetric biosensing. Angew. Chem. Int. Ed., 135, e202300119 (2023).