Key interactions with deazariboflavin cofactor for light-driven energy transfer in Xenopus (6–4) photolyase

Abstract

Photolyases are flavoenzymes responsible for light-driven repair of carcinogenic crosslinks formed in DNA by UV exposure. They possess two non-covalently bound chromophores: flavin adenine dinucleotide (FAD) as a catalytic center and an auxiliary antenna chromophore that harvests photons and transfers solar energy to the catalytic center. Although the energy transfer reaction has been characterized by time-resolved spectroscopy, it is strikingly important to understand how well natural biological systems organize the chromophores for the efficient energy transfer. Here, we comprehensively characterized the binding of 8-hydroxy-7,8-didemethyl-5-deazariboflavin (8-HDF) to Xenopus (6–4) photolyase. In silico simulations indicated that a hydrophobic amino acid residue located at the entrance of the binding site dominates translocation of a loop upon binding of 8-HDF, and a mutation of this residue caused dysfunction of the efficient energy transfer in the DNA repair reaction. Mutational analyses of the protein combined with modification of the chromophore suggested that Coulombic interactions between positively charged residues in the protein and the phenoxide moiety in 8-HDF play a key role in accommodation of 8-HDF in the proper direction. This study provides a clear evidence that Xenopus (6–4) photolyase can utilize 8-HDF as the light-harvesting chromophore. The obtained new insights into binding of the natural antenna molecule will be helpful for the development of artificial light-harvesting chromophores and future characterization of the energy transfer in (6–4) photolyase by spectroscopic studies.

Identification and characterization of a prokaryotic 6-4 photolyase from Synechococcus elongatus with a deazariboflavin antenna chromophore

“…The homologous residues in Se PhrB might also interact with the corresponding moieties of 8-HDF. Lys47 is located at ‘left wall’ of the binding pocket (Figure 6B , orange sticks), where a similar basic residue Arg51 was observed in Se PhrA, and Arg51 in Xenopus laevis eukaryotic 6–4 photolyase ( Xl 64), which is crucial for the binding of 8-HDF by forming a salt bridge to the 8-oxy group of the cofactor ( 15 , 27 , 43 ). The role of Lys47 in Af PhrB is not understood.…”

“…This was reminiscent of a recently report that during the red-light photoreduction of Chlamydomonas reinhardtii aCRY (a bifunctional cryptochrome) with 8-HDF from the neutral radical state to the fully reduced state, a positive peak at 458 nm was observed in the illuminated minus non-illuminated difference spectra, which was interpreted as a result of 8-HDF deprotonation ( 42 ). It was reported that protein binding environments and the protonation states of 8-HDF affect its absorption maximum and intensity ( 43 ). Therefore, the negative band at 459 nm might reflect subtle changes in the protein binding environments and the protonation states of 8-HDF upon illumination.…”

Pushing the limits of flash photolysis to unravel the secrets of biological electron and proton transfer

“…We are happy to announce that today, this virtual volume unites 18 contributions by scientists from 17 countries (Canada, Czech republic, Finland, France, Germany, Hungary, India, Italy, Japan, Lebanon, Poland, Russia, Spain, Switzerland, Taiwan, UK, US) touching upon the wide variety of the subjects dealt with by Klaus at various times [25,[127][128][129][130][131][132][133][134][135][136][137][138][139][140][141][142][143].…”

“…Finally, three papers are dedicated to the study of the binding of redox-active cofactors (flavins and quinones) and how they impact protein function [141][142][143]. Morimoto et al used both nanosecond molecular dynamics simulations and a repair essay on a minute timescale to assess the role of the protein in binding and functioning of the antenna cofactor 8-HDF in Xenopus (6-4) photolyase.…”

“…Morimoto et al used both nanosecond molecular dynamics simulations and a repair essay on a minute timescale to assess the role of the protein in binding and functioning of the antenna cofactor 8-HDF in Xenopus (6-4) photolyase. In silico simulations indicated that a hydrophobic amino acid residue located at the entrance of the binding site dominates translocation of a loop upon binding of 8-HDF, and a mutation of this residue caused dysfunction of the efficient energy transfer in the DNA repair reaction [141]. Lafaye et al modified the cofactor binding specificity of the engineered flavoprotein miniSOG to enhance the production yield of singlet oxygen ΦΔ.…”