Photoinduced damage of AsLOV2 domain is accompanied by increased singlet oxygen production due to flavin dissociation

Abstract

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 .

Photodynamic Activation of Cholecystokinin 1 Receptor with Different Genetically Encoded Protein Photosensitizers and from Varied Subcellular Sites

“…The reason for the satisfactory performance by miniSOG with a ϕ 1 O 2 of only 0.03 to permanently activated photodynamically CCK1R in miniSOG PM -AR4-2J cells in the present work might be related to the progressive photochemical transformation of the fluorophore flavin mononucleotide (FMN) to lumichrome and the photo-oxidization of internal residues in miniSOG to significantly increase its ϕ 1 O 2 up to 10-fold [ 72 ]. In this regard, it is interesting to note that with AsLOV2 (not studied in the present work, and its ϕ 1 O 2 is not determined; see Table A1 ), the LOV2 domain of Avena sativa phototropin 1, light irradiation was found to induce progressive photochemical dissociation or the release of FMN from the AsLOV2 protein moiety, leading to significantly increased ϕ 1 O 2 [ 59 ].…”

“…A red fluorescent protein (TagRFP) for tagging larger proteins has been found to show a noted 1 O 2 quantum yield [ 54 ]. 1 O 2 -generating flavin-binding fluorescent protein (FbFP) photosensitizers originally from other source organisms, such as Pp2FbFP (from Pseudomonas putida ), DsFbFp (from Dinoroseobacter shibae ), EcFbFP (from Bacillus subtilis ), CreiLOV (from Chlamydomonas reinhardtii ), Mr4511 C71G (from Methylobacterium radiotolerans ), and AsLOV2 (from Aveva sativa ), have also appeared, all with desirably sufficient 1 O 2 quantum yields [ 55 , 56 , 57 , 58 , 59 ] (for a list of all GEPP that have appeared in the literature so far, see Table A1 ).…”

Spheroidal Model of SKBR3 and U87MG Cancer Cells for Live Imaging of Caspase-3 during Apoptosis Induced by Singlet Oxygen in Photodynamic Therapy

“…The optical setup was described in our previous work [ 25 ]. A laser system consisting of a pulsed optical parametric oscillator (OPO) (GWU basiScan-M, Erfstadt, Germany) pumped with the third harmonic of a Nd:YAG laser (Spectra-Physics, Quanta-Ray, INDI-HG-10S, Milpitas, CA, USA) was used to excite the samples.…”

Singlet oxygen quenching as a probe for cytochrome c molten globule state formation

“…The same experimental setup as in our previous studies was used. 1,27 In these studies, the samples were irradiated by 5-7 ns-long laser pulses from an Nd:YAG OPO system operated at 10 Hz and tuned to 475 nm. The average laser power on the sample was set to 130 mW.…”