A combination of covalent labeling techniques and mass spectrometry (MS) is currently a progressive approach for deriving insights related to the mapping of protein surfaces or protein–ligand interactions. In this study, we mapped an interaction interface between the DNA binding domain (DBD) of FOXO4 protein and the DNA binding element (DAF16) using fast photochemical oxidation of proteins (FPOP). Residues involved in protein–DNA interaction were identified using the bottom-up approach. To confirm the findings and avoid a misinterpretation of the obtained data, caused by possible multiple radical oxidations leading to the protein surface alteration and oxidation of deeply buried amino acid residues, a top-down approach was employed for the first time in FPOP analysis. An isolation of singly oxidized ions enabled their gas-phase separation from multiply oxidized species followed by CID and ECD fragmentation. Application of both fragmentation techniques allowed generation of complementary fragment sets, out of which the regions shielded in the presence of DNA were deduced. The findings obtained by bottom-up and top-down approaches were highly consistent. Finally, FPOP results were compared with those of the HDX study of the FOXO4-DBD·DAF16 complex. No contradictions were found between the methods. Moreover, their combination provides complementary information related to the structure and dynamics of the protein–DNA complex. Data are available via ProteomeXchange with identifier PXD027624.

Flavin mononucleotide (FMN) belongs to the group of very efficient endogenous photosensitizers producing singlet oxygen, 1 o 2 , but with limited ability to be targeted. On the other hand, in geneticallyencoded photosensitizers, which can be targeted by means of various tags, the efficiency of FMN to produce 1 o 2 is significantly diminished due to its interactions with surrounding amino acid residues. Recently, an increase of 1 o 2 production yield by FMN buried in a protein matrix was achieved by a decrease of quenching of the cofactor excited states by weakening of the protein-FMN interactions while still forming a complex. Here, we suggest an alternative approach which relies on the blue light irradiation-induced dissociation of FMN to solvent. This dissociation unlocks the full capacity of FMN as 1 o 2 producer. Our suggestion is based on the study of an irradiation effect on two variants of the LOV2 domain from Avena sativa; wild type, AsLOV2 wt, and the variant with a replaced cysteine residue, AsLOV2 C450A. We detected irradiation-induced conformational changes as well as oxidation of several amino acids in both AsLOV2 variants. Detailed analysis of these observations indicates that irradiationinduced increase in 1 o 2 production is caused by a release of FMN from the protein. Moreover, an increased FMN dissociation from AsLOV2 wt in comparison with AsLOV2 C450A points to a role of C450 oxidation in repelling the cofactor from the protein. Flavin mononucleotide (FMN) belongs to a group of efficient endogenous photosensitizers in cells with rather high singlet oxygen, 1 O 2 , quantum yield (Φ Δ) within the range 0.51-0.65 1,2. Depending on FMN concentrations and concentrations of available oxygen, the flavin(s) can be even more effective 1 O 2 generators than exogenous porphyrins used for cell killing in photodynamic therapy (PDT). To minimize the potential deleterious effect of flavins to cells, the isoalloxazine moiety of flavin cofactors is typically deeply buried in the protein core of flavoenzymes 3 or storage proteins 4. Singlet oxygen, the lowest energy excited electronic state of molecular oxygen, belongs to the group of reactive oxygen species (ROS), which includes superoxide anion (O 2 •−), hydrogen peroxide (H 2 O 2), and hydroxyl radical (HO •), enabling to oxidize and/or oxygenate many biologically relevant molecules 5,6. Singlet oxygen can be produced in a variety of ways by physical mechanisms, including energy transfer from the excited triplet states of particular chromophores to molecular oxygen 7 , or by chemical mechanisms as one of the products of peroxidase enzymes 8. In biological systems, 1 O 2 is usually generated by electronic energy transfer from an excited state of a photosensitive molecule, so-called photosensitizer (PS), to ground state O 2 6. The high reactivity of singlet oxygen towards biological molecules is relevant in the context of PDT 9 and chromophore-assisted laser inactivation (CALI) of proteins and cells 10,11 .

An efficient way to generate [(L)CuO] complexes with a number of monodentate and bidentate ligands (L) from their [(L)Cu(ClO )] precursors by electrospray ionization was herein explored. Further, we studied [(L)CuO] with L=9,10-phenanthraquinone, 1,10-phenanthroline, and acetonitrile in detail. The signature of these terminal copper-oxo complexes was found to be elimination of the oxygen atom upon collisional activation. We investigated and compared their reactions with water, ethane, ethylene, and 1,4-cyclohexadiene. The [(MeCN)CuO] complex oxidized water and performed C-H activation and hydroxylation of ethane. The complexes with bidentate ligands did not react with water and oxidized only larger hydrocarbons. All the investigated complexes showed comparable reactivities in the oxygen-transfer reaction with ethylene.

A new heterogeneous acidic catalyst was successfully prepared by impregnation of silica (Aerosil 300) by an acidic ionic liquid, named 1-(4-sulfonic acid)butylpyridinium hydrogen sulfate [PYC 4 SO 3 H][HSO 4 ], and characterized using FT-IR spectroscopy, the N 2 adsorption/desorption analysis (BET), thermal analysis (TG/ DTG), and X-ray diffraction (XRD) techniques. The amount of loaded acidic ionic liquid on Aerosil 300 support was determined by acid-base titration. This new solid acidic supported heterogeneous catalyst exhibits excellent activity in the synthesis of 2-aryl-2,3-dihydroquinazolin-4(1H)-ones by cyclocondensation reaction of 2-aminobenzamide with aromatic aldehydes under solvent-free conditions and the desired products were obtained in very short reaction times with high yields. This catalyst has the advantages of an easy catalyst separation from the reaction medium and lower problems of corrosion. Recycling of the catalyst and avoidance of using harmful organic solvent are other advantages of this simple procedure.