Thin films of nanostructured hematite (α-Fe2O3) and carbon quantum dots-incorporated hematite (CQDs@α-Fe2O3) were uniformly grown on titanium substrate and their antibacterial properties against Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria were studied under dark and illumination conditions. The surface morphology of the samples was investigated with FE-SEM and HRTEM. The antimicrobial investigations demonstrate that α-Fe2O3 and CQDs@α-Fe2O3 are toxic to the selected microorganisms and the samples exhibit sustainable antibacterial activity against Gram-positive bacterial strains compared to Gram-negative bacteria mainly due to the presence of extra outer membrane layer in Gram negative bacteria. Based on the results of the antibacterial activity of α-Fe2O3 and CQDs@α-Fe2O3 under dark and the light irradiation conditions, it was concluded that the preferred mechanism of bactericidal activity of hematitebased thin films is both the penetration of iron cations into the bacteria cell via their membrane and the generation of reactive oxygen species. These results indicate that CQDs@α-Fe2O3 nanoparticulates provide insight into the development of visible-light antimicrobial materials for their potential bactericidal applications.

The enzyme nicotine oxidoreductase (NicA2) is a member of the flavoprotein amine oxidase family that uses a cytochrome c protein (CycN) as its oxidant instead of dioxygen, which is the oxidant used by most other members of this enzyme family. We recently identified a potential binding site for CycN on the surface of NicA2 through rigid body docking [J. Biol. Chem. 2022, 298 (8), 102251]. However, this potential binding interface has not been experimentally validated. In this paper, we used unnatural amino acid incorporation to probe the binding interface between NicA2 and CycN. Our results are consistent with a structural model of the NicA2-CycN complex predicted by protein–protein docking and AlphaFold, suggesting that this is the binding site for CycN on NicA2’s surface. Based on additional mutagenesis of potentially redox active residues in NicA2, we propose that electron transfer from NicA2’s flavin to CycN’s heme occurs without the assistance of a protein-derived wire.

RationalePurification of recombinant proteins is a necessary step for functional or structural studies and other applications. Immobilized metal affinity chromatography is a common recombinant protein purification method. Mass spectrometry (MS) allows for confirmation of identity of expressed proteins and unambiguous detection of enzymatic substrates and reaction products. We demonstrate the detection of enzymes purified on immobilized metal affinity surfaces by direct or ambient ionization MS, and follow their enzymatic reactions by direct electrospray ionization (ESI) or desorption electrospray ionization (DESI).MethodsA protein standard, His‐Ubq, and two recombinant proteins, His‐SHAN and His‐CS, expressed in Escherichia coli were immobilized on two immobilized metal affinity systems, Cu–nitriloacetic acid (Cu‐NTA) and Ni‐NTA. The proteins were purified on surface, and released in the ESI spray solvent for direct infusion, when using the 96‐well plate form factor, or analyzed directly from immobilized metal affinity‐coated microscope slides by DESI‐MS. Enzyme activity was followed by incubating the substrates in wells or by depositing substrate on immobilized protein on coated slides for analysis.ResultsSmall proteins (His‐Ubq) and medium proteins (His‐SAHN) could readily be detected from 96‐well plates by direct infusion ESI, or from microscope slides by DESI‐MS after purification on surface from clarified E. coli cell lysate. Protein oxidation was observed for immobilized proteins on both Cu‐NTA and Ni‐NTA; however, this did not hamper the enzymatic reactions of these proteins. Both the nucleosidase reaction products for His‐SAHN and the methylation product of His‐CS (theobromine to caffeine) were detected.ConclusionsThe immobilization, purification, release and detection of His‐tagged recombinant proteins using immobilized metal affinity surfaces for direct infusion ESI‐MS or ambient DESI‐MS analyses were successfully demonstrated. Recombinant proteins were purified to allow identification directly out of clarified cell lysate. Biological activities of the recombinant proteins were preserved allowing the investigation of enzymatic activity via MS.