Limited solvation of an electron donating tryptophan stabilizes a photoinduced charge-separated state in plant (6–4) photolyase

Abstract

Abstract(6–4) Photolyases ((6–4) PLs) are ubiquitous photoenzymes that use the energy of sunlight to catalyze the repair of carcinogenic UV-induced DNA lesions, pyrimidine(6–4)pyrimidone photoproducts. To repair DNA, (6–4) PLs must first undergo so-called photoactivation, in which their excited flavin adenine dinucleotide (FAD) cofactor is reduced in one or two steps to catalytically active FADH− via a chain of three or four conserved tryptophan residues, transiently forming FAD•−/FADH− ⋯ TrpH•+ pairs separated by distances of 15 to 20 Å. Photolyases and related photoreceptors cryptochromes use a plethora of tricks to prevent charge recombination of photoinduced donor–acceptor pairs, such as chain branching and elongation, rapid deprotonation of TrpH•+ or protonation of FAD•−. Here, we address Arabidopsis thaliana (6–4) PL (At64) photoactivation by combining molecular biology, in vivo survival assays, static and time-resolved spectroscopy and computational methods. We conclude that At64 photoactivation is astonishingly efficient compared to related proteins—due to two factors: exceptionally low losses of photoinduced radical pairs through ultrafast recombination and prevention of solvent access to the terminal Trp3H•+, which significantly extends its lifetime. We propose that a highly conserved histidine residue adjacent to the 3rd Trp plays a key role in Trp3H•+ stabilization.

A deazariboflavin chromophore kinetically stabilizes reduced FAD state in a bifunctional cryptochrome

“…In this study, we produced recombinant At 64, At 64-ΔExLoop, Xl 64, and Cr aCRY using the pET28a(+) plasmid system. At 64- and Xl 64-expressing plasmids were constructed, as shown in the previous study 39 . The plasmid producing the Cr aCRY protein, which lacks the C-terminal tail (497–595) 27 was kindly provided by Prof. Lars-Oliver Essen.…”

“…To prepare At 64 and Xl 64, E. coli C41 (DE3)/pLysS (Lucigen) cells were transformed with the pET-28a(+) plasmid encoding At 64 or Xl 64 gene, and protein production and purification were performed as previously described 31 , 39 . Preparation of At 64-ΔExLoop was performed using the same procedure as that used for At 64.…”

“…To monitor reoxidation kinetics, FAD in a protein was photoreduced under anaerobic conditions as previously described 39 . An anaerobic sample containing ~ 5 µM protein and 5 mM L -cysteine in a reaction buffer (20 mM phosphate, 500 mM NaCl, 10% glycerol, pH 7.5), which was prepared in the cuvette used for the midpoint potential measurement, was illuminated with continuous light (430–800 nm) from a MAX-150 xenon lamp (Asahi Spectra) through a 10 × 8 mm window of the cuvette on ice.…”

Insights into Molecular Structure of Pterins Suitable for Biomedical Applications

“…The flavin radical can also be formed as a result of the oxidation of the photoexcited flavin dihydroform; this reaction is the basis for the functioning of DNA photolyases (see below) [ 221 , 222 ]. Additional stability of the flavin radical can be imparted by amino acids that surround the flavin in the reaction center of the protein [ 223 , 224 ].…”