An unexpected all-metal aromatic tetranuclear silver cluster in human copper chaperone Atox1

Metal clusters, such as iron–sulfur clusters, play key roles in sustaining life and are intimately involved in the functions of metalloproteins. Herein we report the formation and crystal structure of a planar square tetranuclear silver cluster when silver ions were mixed with human copper chaperone Atox1. Quantum chemical studies reveal that two Ag 5s1 electrons in the tetranuclear silver cluster fully occupy the one bonding molecular orbital, with the assumption that this Ag4 cluster is Ag42+, leading to extensive electron delocalization over the planar square and significant stabilization. This bonding pattern of the tetranuclear silver cluster represents an aromatic all-metal structure that follows a 4n + 2 electron counting rule (n = 0). This is the first time an all-metal aromatic silver cluster was observed in a protein.

protease at 277 K overnight. Ni-affinity chromatography (His Trap HP) was used again to obtain Atox1, which was separated from the 6×His -TEV fusion protein. High purity Atox1 was obtained after further purified by size-exclusion chromatography (Superdex 30) and was concentrated. with a ProSwift™ RP-3U LC Column (4.6×50 mm, SS, Thermo Scientific™). Positive ion Electrospray Ionization (ESI) mass spectra for intact protein were obtained with an Agilent 6224 mass spectrometer equipped with an ESI interface and a time-of-flight (TOF) mass detector (Agilent Technologies). Mass spectra were analyzed and deconvoluted using an Agilent software MassHunter version B.04.00 (Agilent Technologies).

Inductively coupled plasma mass spectrometry (ICP-MS) experiments.
To confirm the binding condition of Atox1 fusion protein and Ag, we conducted ICP-MS detection in a 220 μl complex solution containing Atox1 fusion protein and Ag. As a result, we found that the molar ratio of Ag (0.278 × 10 -6 mol) and Atox1 (0.064 × 10 -6 mol) was about 4.34 in Ag-Atox1 complex.

Single-molecule AFM experiments.
Single-molecule AFM experiments were carried out on a commercial AFM (Force Robot 300, JPK, Berlin, Germany). All the force-extension experiments were carried out in Tris-HCl buffer (25mM Tris, 72mM NaCl). Protein sample (0.1mg ml -1 ,150μl) was directly deposited on a freshly cleaved glass surface for 2h and was washed with buffer to remove unreacted protein. We modified the cantilever tip with cys-Xmod-doc. The gold-coated cantilever was immersed in a protein solution (0.1 mg ml -1 ) for 1 hour at room temperature to allow the formation of goldthiol linkage. The physically adsorbed proteins were removed by rinsing the cantilever tip with deionized water for at least 5 times in an incubator. Then, the sample chamber was filled with 1ml buffer before the measurement. The spring constant of the AFM cantilevers (Biolever-RC-150VB-70 from Olympus) was calibrated using the equipartition theorem before each experiment, with a typical value of 6pN nm -1 . The pulling speed was 400 nm s -1 for all traces.
EPR measurement. 0.2 mM Atox1 was incubated with 10 molar equivalents of AgNO3 in 20 mM Tris buffer containing 200 mM NaCl. Then the spin-trapping agent 5,5-dimethylpyrroline-N-oxide (DMPO) was added to the reaction mixture.
After shaking for 1 minute, the sample was transferred to a quartz capillary tube. For MTSSL labeling, 0.2 mM Atox1 was incubated with 10 molar equivalents of MTSSL overnight at 4°C. Then the reaction mixture was added to Ni-NTA to remove free spin label. The continuous-wave electron paramagnetic resonance (CW-EPR) spectra were recorded on a Bruker A300 spectrometer (Bruker Biospin GmbH, Rheinstetten, Germany) at X-band (9.5 GHz).  Collaborative Computational Project, Number 4, 1994) and manual improvement in Coot. All structural representations were generated using PyMOL (DeLano, 2002) with subsequent ray tracing.

Theoretical Analyses and Computational Modeling.
In Figure 3, the electronic structure of Ag 4 2+ cluster based on the experimentally measured geometry ( Figure   2) have been investigated using density functional theory (DFT) with Amsterdam Density Functional 2019 program.
The calculations were done using PBE exchange-correlation functional and the TZP Slater basis sets. Frozen core approximation was applied to the [1s 2 …4p 6 ] core of Ag, and Zeroth-Order Regular Approximation to the Dirac Equation (ZORA) was used to account for the scalar relativistic (SR) effect.
The constrained DFT geometry optimization was done at the level of SR-ZORA PBE/DZP with Grimme D3-BJ Dispersion Correction. The model of constrained geometry optimization adopted [Ag 4 ] q+ cluster inside the frozen experimental cavity, with size about 5 Å around the [Ag 4 ] q+ core. The calculated molecular orbital (MO) energy levels and wavefunctions of Ag 4 2+ cluster are listed in Table S3-S5, which were analyzed using Hückel method based on three kinds of geometry structures with T d , D 4h and D 2h symmetry. The MO energies and frontier MOs of optimized geometry structures with T d , D 4h and D 2h symmetries and experimentally measured structure with C 2 symmetry were studied by ADF 2019 program with level of PBE/TZP, frozen core approximation and ZORA scalar relativistic method.
The ELF color-filled map and multicenter bond index were calculated using Gaussian-16B and Multiwfn-3.8 at the level of PBE0/def2-TZVP. The nucleus-independent chemical shift (NICS) of Ag 4 2+ cluster based on the experimentally measured geometry was studied using Multiwfn and Gaussian with level of B3LYP/def2-SVP.