Atomic-Level Engineering of Protein-Templated Gold Clusterzymes with Tunable Fluorescence and Multi-Enzyme Activities for Antiaging Treatment

Atomic precision synthesis and modulation of nanozymes are critical for evaluating their performance and developing functional applications. Herein, we developed an in situ strategy for the controlled synthesis of gold clusterzymes (AuCEs) within the confined cavities of the protein scaffolds. Using a stable dodecameric protein (SP1) as a ligand, we fused a short peptide containing the CCY sequence (SP1-CCY) to the N-terminus of SP1, which is located within its cyclic structure. In this design, cysteine and tyrosine synergistically chelate and reduce Au ions, while the confined protein structure restricts cluster growth, leading to the formation of a 25-gold atom (Au 25 ) nanocluster with strong fluorescence and peroxidase-like activity. Unlike chemical ligands, protein scaffolds offer atomic-level precision and allow for site-specific modulation of the local environment through rational mutations, such as L9D, L9I, and E73I/L9I. This allows precise control of both fluorescence and enzymatic activity via protein engineering. Additionally, heteroatom doping of the Au core with copper endows the AuCEs with significant superoxide dismutase-like activity. Antioxidant assays demonstrate that the optimized AuCEs exhibit potent reactive oxygen species scavenging activity, providing significant protection to Caenorhabditis elegans from oxidative stress and highlighting their potential as therapeutic candidates for aging-related diseases.