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

Martina Petrenčáková,Pavol Miškovský,Petr Man,Ghazaleh Yassaghi,Marc-Simon Schwer,František Filandr,Erik Sedlák,Tibor Kožár,Petr Novák,Daniel Jancura,Andrej Hovan,Gregor Bánó,Andreas Plückthun

doi:10.1038/s41598-020-60861-2

Abstract

Flavin mononucleotide (FMN) belongs to the group of very efficient endogenous photosensitizers producing singlet oxygen, 1O2, but with limited ability to be targeted. On the other hand, in genetically-encoded photosensitizers, which can be targeted by means of various tags, the efficiency of FMN to produce 1O2 is significantly diminished due to its interactions with surrounding amino acid residues. Recently, an increase of 1O2 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 1O2 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 irradiation-induced increase in 1O2 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.

Highlights

On the other hand, the improved control regarding 1O2 production by using genetically-encoded photosensitizers leads to attempts to utilize them as antimicrobial agents[15,16] or in PDT17
We conclude that the irradiation-induced increase of 1O2 production in the AsLOV2 variants is due to a release of Flavin mononucleotide (FMN) to solvent as a result of oxidative modification of certain amino acids in the AsLOV2 structure
Based on the experimental observation, we proposed a model that schematically describes irradiation-induced changes in AsLOV2 C450A accompanied by FMN release (Scheme 1)

Summary

Introduction

The improved control regarding 1O2 production by using genetically-encoded photosensitizers leads to attempts to utilize them as antimicrobial agents[15,16] or in PDT17. Efficiency of the 1O2 production in miniSOG (mini-singlet oxygen generator; ΦΔ = 0.03–0.05)[21,26], engineered from the FMN-containing LOV2 domain of Arabidopsis thaliana phototropin 227, upon chemical denaturation increased over 10-fold in comparison with its native form[21] These observations led to efforts to develop protein PSs with improved 1O2 production, such as SOPP (singlet oxygen photosensitizing protein; ΦΔ = 0.19–0.26)[26] and SOPP3 with ΦΔ = 0.60, comparable to that of free FMN23. These improved variants of miniSOG were obtained by identification and replacement of amino acids responsible for: (i) steric barriers for oxygen diffusion toward the PS, (ii) quenching of FMN triplet state by electron transfer, and (iii) quenching of produced 1O2 by chemical reactions[23,26]. Our results suggest a new approach towards designing an efficient protein photosensitizer as a carrier of a chromophore that can be subsequently released by irradiation of the protein at the site of its action

Methods

Results

Discussion

Conclusion