**4. Conclusions**

The present work demonstrates that a redox process that leads to an admixture of Au(0), Au(I), and Au(II) is taking place upon entrapment of Au(III) in BSA and several Au atoms of AuNCs are directly interacting through Cys and Tyr residues with BSA. The authors observed that Tyr molecules are able to reduce Au(III) to Au(0) in the form of AuNPs, while Cys did not provide the electron equivalents needed for the Au(III) reduction. It is also demonstrated that UV irradiation induces irreversible damages in AuBSA and radical species are generated during photoexcitation in the protein backbone. Since there is no evidence of electronic communication between AuNCs and BSA according to the LEPR experiments (in situ off-on-off cycles under UV light, at 90 K), any energy and/or charge transfer can be excluded as a key mechanism for expressing the bright fluorescence phenomenon observed, with maximum emission at 655 nm, in AuBSA. Furthermore, evidence was given for the in situ growth of AuNCs to AuNPs and migration of Au nanostructures during HR-TEM and EDS data acquisition. This work further reinforces the knowledge that results obtained by using sophisticated techniques exploiting electrons and/or X-ray irradiation in the investigation of gold nanostructures embedded in protein matrixes must be considered carefully, because the experimental evidence might be severely hampered by induced radiation damages on the protein structure.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/nano12091425/s1, Figure S1: Corrected emission spectrum of AuBSA in deionized water; Table S1: Quantum yield and wavelength of maximum fluorescence of AuBSA; Figure S2. CW-EPR spectrum obtained after 20 min of UV irradiation of BSA in water. Experimental parameters: T = 90 K, 9.0802 GHz frequency, 100 kHz modulation frequency, 0.03 s time constant, 1.60 mW applied power, 0.5 mT modulation width, 6 × 100 Gain, 1 min sweep time, and 5 scans accumulated and averaged. The simulation of the various spin components is shown together with their sum (red-line); Figure S3. Extinction spectra of Au-Tyr in deionized water 1 min and 45 s after synthesis (grey line) and 61 min and 45 s after synthesis (wine line). (B) and (C) TEM images of Au-Tyr recorded at different magnifications; Figure S4. CW-EPR spectrum of AuBSA in water recorded at T = 90 K in dark conditions with the EPR simulation of the diverse spin-components associated to the Au nanoclusters; Figure S5: Further STEM images of AuBSA; Figure S6: Particle size distributions derived from TEM, STEM and HR-TEM images; Figure S7: Lattice spaces determined for several AuNCs of AuBSA in a particular HR-TEM image.

**Author Contributions:** Conceptualization: K.Š. and G.Z.; data curation: G.Z., R.O., O.T. and D.P.; formal analysis: G.Z., R.O., O.T. and D.P.; funding acquisition: K.Š. and V.F.; investigation: G.Z., R.O., O.T. and D.P.; methodology: G.Z. and K.Š.; project administration: K.Š.; supervision: K.Š.; validation: G.Z., R.O., O.T. and D.P.; visualization: G.Z., R.O., O.T. and D.P.; writing—original draft: K.Š.; writing—review and editing: G.Z. and K.Š. All authors have read and agreed to the published version of the manuscript.

**Funding:** K.Š. thanks, for financial support, the Grant Agency of the Czech Republic (grant no. 19-03207S). K.Š. and R.O. thank the Internal Grant Agency of Palacký University (grant numbers IGA\_PrF\_2021\_003 and IGA\_PrF\_2022\_003). D.P. and V.F. acknowledge the MEYS CR (project no. LM2018129 Czech BioImaging) and ERDF projects (CZ.02.1.01/0.0/0.0/18\_046/0016045, CZ.02.1.01/0.0/0.0/16\_013/0001775) for

funding. G.Z. and O.T. thank the ERDF/ESF project "Nano4Future" (project no. CZ.02.1.01/0.0/0.0/1 6\_019/0000754).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** Monika Opatíková is acknowledged for TEM imaging of Au-Tyr system and Erik Dostál is thanked for this and Au-Cys system preparation and UV-vis extinction measurements.

**Conflicts of Interest:** The authors declare no conflict of interest.
