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![Spectroscopic analysis of cofactor binding and relative repair efficiency of the (6-4) photolyases. (A) UV and fluorescence spectra of the reduced (6-4) DNA photolyase from D. melanogaster (10 mM Na-dithionite) before and after reconstitution with F 0. (B) Relative repair activity of the (6-4) photolyases from D. melanogaster and O. tauri in the absence and presence of F 0 [cDNA 100 mol/L, c(6-4)photolyase 20 mol/L in buffer A; 50 mM TrisHCl, pH 7.5; 100 mM NaCl; 5 mM DTT; 1 mM EDTA; and 5% glycerine] (9).](profile/Thomas-Carell/publication/42376722/figure/fig2/AS:394351462109186@1471032123331/Spectroscopic-analysis-of-cofactor-binding-and-relative-repair-efficiency-of-the-6-4.png)
Spectroscopic analysis of cofactor binding and relative repair efficiency of the (6-4) photolyases. (A) UV and fluorescence spectra of the reduced (6-4) DNA photolyase from D. melanogaster (10 mM Na-dithionite) before and after reconstitution with F 0. (B) Relative repair activity of the (6-4) photolyases from D. melanogaster and O. tauri in the absence and presence of F 0 [cDNA 100 mol/L, c(6-4)photolyase 20 mol/L in buffer A; 50 mM TrisHCl, pH 7.5; 100 mM NaCl; 5 mM DTT; 1 mM EDTA; and 5% glycerine] (9).
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Archae possess unique biochemical systems quite distinct from the pathways present in eukaryotes and eubacteria. 7,8−Dimethyl−8−hydroxy−5deazaflavin (F0) and F420 are unique deazaflavin−containing coenzyme and methanogenic signature molecules, essential for a variety of biochemical transformations associated with methane biosynthesis and light−depe...
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... the lack of binding. In all cases, only an empty photoan- tenna-binding site was detected. Finally, we added the archaeal cofactor F 0 to the eukaryotic D. melanogaster (6-4) photolyase and noticed an immediate change of the absorption and fluo- rescence spectrum. After addition of F 0 , an additional absorp- tion peak at (abs) max 440 nm ( Fig. 2A Left) and a strong fluorescence signal at (em) max 475 nm (Fig. 2 A Right) were detected. These data are in perfect agreement with the spec- troscopic signatures of F 0 containing DNA photolyases from cyanobacteria. They show that F 0 is not only bound but also correctly deprotonated in the chromophore-binding pocket (10,11), because the ...
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... site was detected. Finally, we added the archaeal cofactor F 0 to the eukaryotic D. melanogaster (6-4) photolyase and noticed an immediate change of the absorption and fluo- rescence spectrum. After addition of F 0 , an additional absorp- tion peak at (abs) max 440 nm ( Fig. 2A Left) and a strong fluorescence signal at (em) max 475 nm (Fig. 2 A Right) were detected. These data are in perfect agreement with the spec- troscopic signatures of F 0 containing DNA photolyases from cyanobacteria. They show that F 0 is not only bound but also correctly deprotonated in the chromophore-binding pocket (10,11), because the redox-active, protonated version of F 0 has clearly distinguishable ...
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... investigate whether the enzyme containing the deproto- nated F 0 is able to funnel light energy to the FADH cofactor, we compared the enzymatic repair activity of F 0 -deficient en- zyme with F 0 -reconstituted D. melanogaster (6-4) photolyase at 440 nm (Fig. 2B). In the presence of F 0 , the repair efficiency was increased significantly, by approximately a factor of 5. This shows that the F 0 cofactor is not only bound and deprotonated by the enzymes, but that it is able to funnel energy to the FADH . As reported previously, the antenna chromophore is not essential for DNA binding and ...
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... overall structure with the F 0 cofactor tightly bound in the light-harvesting pocket is depicted in Fig. 3A (see Fig. S2 for an example of the electron density). Deprotonation of F 0 is achieved by the residues Arg-60 and Lys-266, which contact the 8-hydroxy group (Fig. 3B). Moreover, a conformational change of the surface loop consisting of residues 47-65 was observed, with Ile-50 packing over the deazaflavin ring system and Trp-53 partly shielding the ...
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... could be observed, in line with the data of the F 0 -reconstituted (6-4) photolyase from D. melano- gaster. Determination of the repair activity before and after reconstitution revealed again that binding of F 0 increases the repair efficiency by a factor of 5, showing that this (6-4) photolyase also used the F 0 cofactor as a light antenna. (Fig. 2B). In O. tauri, a BLAST search provided evidence for the presence of the fbiC gene, coding for the bifunctional F 0 synthetase (Figs. S4 and S5) and initially characterized in Mycobacterium tuber- culosis (25). FbiC belongs to the radical S-Adenosyl methionine (SAM) superfamily of enzymes and catalyzes the synthesis of F 0 from ...
Citations
... Because the pocket is known to be a mutational hotspot co-evolving with protein functions in animal CRYs 35 , the structural diversity in the secondary pocket among (6-4)PP-repairing proteins may be related to the kinetic variations of the reoxidation process. Regarding (6-4) PP-repairing proteins, Xl64 and CraCRY can harbor 8-hydroxy-7,8-didemethyl-5-deazariboflavin (8-HDF) as the antenna chromophore, transferring its absorbed energy to FAD 10,27 . However, a previous study 12 suggested that At64 would completely lose its ability to bind 8-HDF, presumably because of the exclusive presence of an extended loop near the secondary pocket (Fig. 3a). ...
An animal-like cryptochrome derived from Chlamydomonas reinhardtii (CraCRY) is a bifunctional flavoenzyme harboring flavin adenine dinucleotide (FAD) as a photoreceptive/catalytic center and functions both in the regulation of gene transcription and the repair of UV-induced DNA lesions in a light-dependent manner, using different FAD redox states. To address how CraCRY stabilizes the physiologically relevant redox state of FAD, we investigated the thermodynamic and kinetic stability of the two-electron reduced anionic FAD state (FADH⁻) in CraCRY and related (6–4) photolyases. The thermodynamic stability of FADH⁻ remained almost the same compared to that of all tested proteins. However, the kinetic stability of FADH⁻ varied remarkably depending on the local structure of the secondary pocket, where an auxiliary chromophore, 8-hydroxy-7,8-didemethyl-5-deazariboflavin (8-HDF), can be accommodated. The observed effect of 8-HDF uptake on the enhancement of the kinetic stability of FADH⁻ suggests an essential role of 8-HDF in the bifunctionality of CraCRY.
... A similar residue (Ile51) is observed in AfPhrB in the homologous position near the lumazine ring of DMRL ( Fig. 3A and 3B). Lys49 in SePhrB contacts the 8-oxo group of 8-HDF, which may facilitate the deprotonation of 8-HDF to form a highly colored delocalized anion state 31 . Interestingly, the residue is also conserved in AfPhrB (Lys47), although it seems to be useless in DMRL binding (Fig. 3A and 3B). ...
Se PhrB provides the first structure of prokaryotic 6 − 4 photolyases with 8-HDF as the antenna cofactor, and also the first structure of photolyases with covalently-linked FAD as the catalytic cofactor. It also contains a [4Fe-4S] cluster coordinated with four conserved cysteine residues. Based on the structural analysis and the inspiration of a study on human primase ²⁷ , we propose that the [4Fe-4S] cluster in Se PhrB may participate in electron transfer and trigger DNA disassociation during catalysis. The binding sites for 8-HDF in Se PhrB and for 8-HDF, DMRL, FMN, or FAD in other photolyases are in homologous positions, which suggests that 8-HDF may be utilized as the antenna cofactor by the last common ancestor of the antenna cofactor-containing photolyases. The formation of the covalent linkage between FAD and Met399 in Se PhrB is light-dependent, which does not require external electron donors. The FAD-methionine photo-adduct in Se PhrB is catalytically proficient and stable under aerobic conditions. The novel findings from Se PhrB suggest that photolyase family is more complex than expected, which warrant further intensive investigation.
... Based on studies by Tamada et al. [32] and Mees et al. [31] and our analysis of the SEL photolyase crystal structure, a total of 59 residues were identified that were proximal (withiñ 2.8 Å) to or interacting with either the bound DNA, or FAD and HDF cofactors. When these residues were compared with corresponding residues in photolyases of Haloarchaea using multiple sequence alignments and structural comparisons, 15 amino acid residues, including six key positions previously reported (R50, G149, E282, N385, D398 and K413) in the SEL photolyase, were found to be changed in at least one of the haloarchaeal photolyase sequences, (see Figure 5b,c) [31,50,51]. ...
Ultraviolet (UV) radiation responses of extremophilic and archaeal microorganisms are of interest from evolutionary, physiological, and astrobiological perspectives. Previous studies determined that the halophilic archaeon, Halobacterium sp. NRC-1, which survives in multiple extremes, is highly tolerant of UV radiation. Here, Halobacterium sp. NRC-1 UV tolerance was compared to taxonomically diverse Haloarchaea isolated from high-elevation salt flats, surface warm and cold hypersaline lakes, and subsurface Permian halite deposits. Haloterrigena/Natrinema spp. from subsurface halite deposits were the least tolerant after exposure to photoreactivating light. This finding was attributed to deviation of amino acid residues in key positions in the DNA photolyase enzyme or to the complete absence of the photolyase gene. Several Halobacterium, Halorubrum and Salarchaeum species from surface environments exposed to high solar irradiance were found to be the most UV tolerant, and Halorubrum lacusprofundi from lake sediment was of intermediate character. These results indicate that high UV tolerance is not a uniform character trait of Haloarchaea and is likely reflective of UV exposure experienced in their environment. This is the first report correlating natural UV tolerance to photolyase gene functionality among Haloarchaea and provides insights into their survival in ancient halite deposits and potentially on the surface of Mars.
... Finally, we confirmed that F 420 binds to MSMEG_2027 with high specificity. F 420 is chemically similar to FMN, flavin adenine dinucleotide (FAD), and F0, a chemical precursor of F 420 consisting of just the core ring without phosphate, lactyl-group and polyglutamate chain [44][45][46]. We tested the response of MSMEG_2027-FGFR1 to these cofactors and found that neither F0, FMN nor FAD inhibited the MAPK/ERK pathway in receptor transfected cells (Figure 4d). ...
Protein-protein interactions (PPIs) mediate many fundamental cellular processes and their control through optically or chemically responsive protein domains has a profound impact on basic research and some clinical applications. Most available chemogenetic methods induce the association, i.e., dimerization or oligomerization, of target proteins, and the few available dissociation approaches either break large oligomeric protein clusters or heteromeric complexes. Here, we have exploited the controlled dissociation of a dimeric oxidoreductase from mycobacteria (MSMEG_2027) by its native cofactor, F420, which is not present in mammals, as a bioorthogonal monomerization switch. We found that in the absence of F420, MSMEG_2027 forms a unique domain-swapped dimer that occludes the cofactor binding site. Substantial remodelling of the intertwined N-terminal helix upon F420 binding results in the dissolution of the dimer. We then show that MSMEG_2027 can be expressed as fusion proteins in human cells and apply it as a tool to induce and release MAPK/ERK signalling downstream of a chimeric fibroblast growth factor receptor 1 (FGFR1) tyrosine kinase. This F420-dependent chemogenetic de-dimerization tool is stoichiometric, based on a single domain and presents a novel mechanism to investigate protein complexes in situ.
... The deazariboflavin cofactor 8-HDF, or named FO, is employed by a number of class I/III CPD photolyases (including SePhrA) (7,39), class II CPD photolyases (27), and eukaryotic 6-4 photolyases/bifunctional cryptochromes (26,(40)(41)(42)(43), to sever as an antenna cofactor that harvests and transfers more light energy to the catalytic FAD cofactor to enhance the photorepair activity of the enzymes. The synthesis of 8-HDF requires a FO synthase that is composed of two subunits CofG and CofH, or a fusion protein FbiC with two domains that are homologous to CofG and CofH (44). ...
... The 8-HDF cofactor is a precursor of F420, which functions analogously to NAD as a two-electron, hydride-transfer coenzyme in a number of archaea and actinomycetes (45,54). Many photolyases also utilize 8-HDF as their antenna cofactor (7,26,27,(39)(40)(41)(42)(43). The DMRL cofactor is an intermediate in the last step of riboflavin biosynthesis (55). ...
Synechococcus elongatus, formerly known as Anacystis nidulans, is a representative species of cyanobacteria. It is also a model organism for the study of photoreactivation, which can be fully photoreactivated even after receiving high UV doses. However, for a long time, only one photolyase was found in S. elongatus that is only able to photorepair UV induced cyclobutane pyrimidine dimers (CPDs) in DNA. Here, we characterize another photolyase in S. elongatus, which belongs to iron-sulfur bacterial cryptochromes and photolyases (FeS-BCP), a subtype of prokaryotic 6-4 photolyases. This photolyase was named SePhrB that could efficiently photorepair 6-4 photoproducts in DNA. Chemical analyses revealed that SePhrB contains a catalytic FAD cofactor and an iron-sulfur cluster. All of previously reported FeS-BCPs contain 6,7-dimethyl-8-ribityllumazine (DMRL) as their antenna chromophores. Here, we first demonstrated that SePhrB possesses 7,8-didemethyl-8-hydroxy-5-deazariboflavin (8-HDF) as an antenna chromophore. Nevertheless, SePhrB could be photoreduced without external electron donors. After being photoreduced, the reduced FAD cofactor in SePhrB was extremely stable against air oxidation. These results suggest that FeS-BCPs are more diverse than expected which deserve further investigation.
... Contrary to the exclusively conserved catalytic chromophore, several types of the auxiliary chromophore are known to function as the light-harvesting chromophore (LHC) in PLs. The examples are 5,10-methenytetrahydrofolate (MTHF) in bacterial PL [17], FAD or flavin mononucleotide (FMN) in thermophilic bacterial PLs [20,21], 6,7-dimethyl-8-ribityllumazine (DMRL) in bacterial PLs [19], and 8-hydroxy-7,8-didemethyl-5-deazariboflavin in class II CPD-PL [22], class I PL in cyanobacteria [23], algae cryptochrome/photolyase families (CPF) [18,24], and insect (6-4)PL [24]. Regardless of the chemical structures of LHCs, their fluorescence overlaps the FADHabsorption, leading to the enhanced light-driven DNA repair via FRET. ...
... Contrary to the exclusively conserved catalytic chromophore, several types of the auxiliary chromophore are known to function as the light-harvesting chromophore (LHC) in PLs. The examples are 5,10-methenytetrahydrofolate (MTHF) in bacterial PL [17], FAD or flavin mononucleotide (FMN) in thermophilic bacterial PLs [20,21], 6,7-dimethyl-8-ribityllumazine (DMRL) in bacterial PLs [19], and 8-hydroxy-7,8-didemethyl-5-deazariboflavin in class II CPD-PL [22], class I PL in cyanobacteria [23], algae cryptochrome/photolyase families (CPF) [18,24], and insect (6-4)PL [24]. Regardless of the chemical structures of LHCs, their fluorescence overlaps the FADHabsorption, leading to the enhanced light-driven DNA repair via FRET. ...
... As the structure of Xl64 has not been reported so far, we used Drosophila melanogaster (6-4)PL (Dm64) structures with or without the 8-HDF cofactor (3CVV [24] and 3CVU [28], referred to as holoDm64 and apoDm64, respectively). Note that Xl64 and Dm64 share 58% identity and 70% homology, and F41, R51, and K256 in Xl64 correspond to I50, R60, and K266 in Dm64, respectively. ...
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.
... In addition to its incorporation into F 420, F O is synthesized independently and its fluorescent properties are exploited by a class of DNA photolyases, which bind F O and FMN as cofactors to mediate the reductive cleavage of DNA pyrimidine dimers (Malhotra et al. 1992;Tamada et al. 1997). F O -utilizing DNA photolyases are present in cyanobacteria, unicellular algae and possibly higher eukaryotes including Drosophila (Mayerl et al. 1990;Sancar 1990;Glas et al. 2009). Like F O , F 420 exhibits analogous autofluorescence and these properties can be used to identify F 420producing organisms such as methanogens and mycobacteria by fluorescence microscopy (Doddema and Vogels 1978;Maglica, Ozdemir and McKinney 2015;Lambrecht et al. 2017), or sort them by flow cytometry. ...
Many bacteria and archaea produce the redox cofactor F420. F420 is structurally similar to the cofactors FAD and FMN but is catalytically more similar to NAD and NADP. These properties allow F420 to catalyze challenging redox reactions, including key steps in methanogenesis, antibiotic biosynthesis, and xenobiotic biodegradation. In the last five years, there has been much progress in understanding its distribution, biosynthesis, role, and applications. Whereas F420 was previously thought to be confined to Actinobacteria and Euryarchaeota, new evidence indicates it is synthesized across the bacterial and archaeal domains, as a result of extensive horizontal and vertical biosynthetic gene transfer. F420 was thought to be synthesized through one biosynthetic pathway; however, recent advances have revealed variants of this pathway and have resolved their key biosynthetic steps. In parallel, new F420-dependent biosynthetic and metabolic processes have been discovered. These advances have enabled the heterologous production of F420 and identified enantioselective F420H2-dependent reductases for biocatalysis. New research has also helped resolve how microorganisms use F420 to influence human and environmental health, providing opportunities for tuberculosis treatment and methane mitigation. Fifty years since its discovery, multiple paradigms associated with F420 have shifted, and new F420-dependent organisms and processes continue to be discovered.
... Contrary to the exclusively conserved FAD chromophore in PLs, the antenna chromophores in PLs are diverse. So far, five natural antenna chromophores with a pteridine architecture have been reported for PLs ( Figure 1B): (i) 5,10-methenyltetrahydrofolate (MTHF) for bacterial (10,11) and algal PLs (12), (ii) flavin mononucleotide (FMN) (13) or (iii) FAD (14) for thermophilic bacterial PLs, (iv) 8-hydroxy-7,8-didemethyl-5-deazariboflavin (8-HDF) for class II CPD-repairing PLs (CPD-PL) (15), Synechococcus elongatus (also known as Anacystis nidulans) PL (16), 6-4PP-repairing PLs ((6-4)PLs) (17), and (v) 6,7-dimethyl-8-ribityllumazine (DMRL) for bacterial PL (18). These molecules are well recognized in the binding site at the cleft between the N-terminal ␣/ domain and the catalytic C-terminal domain in PLs ( Figure 1A) and emit fluorescence that overlaps with the absorption of FADH − , allowing them to transfer the energy presumably via the Förster mechanism (Förster resonance energy transfer, FRET) (19)(20)(21). ...
Photolyases are flavoenzymes responsible for the repair of carcinogenic DNA damage caused by ultraviolet radiation. They harbor the catalytic cofactor flavin adenine dinucleotide (FAD). The light-driven electron transfer from the excited state of the fully-reduced form of FAD to the DNA lesions causes rearrangement of the covalent bonds, leading to the restoration of intact nucleobases. In addition to the catalytic chromophore, some photolyases bear a secondary chromophore with better light absorption capability than FAD, acting as a light-harvesting chromophore that harvests photons in sunlight efficiently and transfers light energy to the catalytic center, as observed in natural photoreceptor proteins. Inspired by nature, we covalently and site-specifically attached a synthetic chromophore to the surface of photolyase using oligonucleotides containing a modified nucleoside and a cyclobutane-type DNA lesion, and successfully enhanced its enzymatic activity in the light-driven DNA repair. Peptide mapping in combination with theoretical calculations identified the amino acid residue that binds to the chromophore, working as an artificial light-harvesting chromophore. Our results broaden the strategies for protein engineering and provide a guideline for tuning of the light perception abilities and enzymatic activity of the photoreceptor proteins.
... The main role of these molecules in DNA photolyases is absorption of light photons and resonance energy transfer through the dipole-dipole interactions onto the catalytic FADH - [2,31,44,45]. Five antenna molecules have been described: (i) MTHF in the majority of eukaryotic and some prokaryotic enzymes (e.g., Escherichia coli, Neurospora crassa, Saccharomyces cerevisiae) [2]; (ii) 8 HDF in some prokaryotes (Streptomyces griseus, Anacystis nidulans, etc.) [2], simplest eukaryotes (Ostreococcus tauri, Scenedesmus acutus) that can synthesize this com pound [46,47], and very seldomly, in eukaryotes that can receive it from the symbiotic bacteria and retain the abil ity to bind deazaflavin (Drosophila melanogaster) [47]; (iii) the second FAD molecule (archaebacterium Sulfolobus tokodaii) [48]; and (iv) FMN (Thermus ther mophilus) [49]. Recently it was shown that (v) 6,7 dimethyl 8 ribityllumazin (DMRL) can function as antenna in prokaryotic (6 4) photolyases (Agrobacterium fabrum, Rhodobacter sphaeroides) [19,50]. ...
... The main role of these molecules in DNA photolyases is absorption of light photons and resonance energy transfer through the dipole-dipole interactions onto the catalytic FADH - [2,31,44,45]. Five antenna molecules have been described: (i) MTHF in the majority of eukaryotic and some prokaryotic enzymes (e.g., Escherichia coli, Neurospora crassa, Saccharomyces cerevisiae) [2]; (ii) 8 HDF in some prokaryotes (Streptomyces griseus, Anacystis nidulans, etc.) [2], simplest eukaryotes (Ostreococcus tauri, Scenedesmus acutus) that can synthesize this com pound [46,47], and very seldomly, in eukaryotes that can receive it from the symbiotic bacteria and retain the abil ity to bind deazaflavin (Drosophila melanogaster) [47]; (iii) the second FAD molecule (archaebacterium Sulfolobus tokodaii) [48]; and (iv) FMN (Thermus ther mophilus) [49]. Recently it was shown that (v) 6,7 dimethyl 8 ribityllumazin (DMRL) can function as antenna in prokaryotic (6 4) photolyases (Agrobacterium fabrum, Rhodobacter sphaeroides) [19,50]. ...
Proteins of the cryptochrome/DNA photolyase family (CPF) are phylogenetically related and structurally conserved flavoproteins that perform various functions. DNA photolyases repair DNA damage caused by UV-B radiation by exposure to UV-A/blue light simultaneously or subsequently. Cryptochromes are photoreceptor proteins regulating circadian clock, morphogenesis, phototaxis, and other responses to UV and blue light in various organisms. The review describes the structure and functions of CPF proteins, their evolutionary relationship, and possible functions of the CPF ancestor protein.
... The deprotonation was rationalized by the presence of positively charged residues lysine 258 (L258) and arginine 55 (R55) next to the 8hydroxyl group 10,29 ( Figure 1C) as well as the red-shifted absorbance maximum at 448 nm, which is similar to spectra previously found for other 8-HDF-binding photolyases. 10,12,34 In these enzymes, the red-shifted absorbance as opposed to the pigment's absorbance maximum in aqueous solution at 421 nm ( Figure 1A) was explained by a negative solvatochromic effect exhibited by charged species in a nonpolar environment such as the protein matrix. 35 Previous experiments investigating the red-light response of aCRY in vitro were conducted exclusively on samples without the antenna pigment 8-HDF. ...
... 12,35 Compared to other photolyases carrying 8-HDF, in aCRY·8-HDF the absorption shift is especially pronounced with a maximum found at 448 nm. 29,36 Only the (6−4) photolyase of Drosophila melanogaster shows a similar shift to the red, 10,36 implying that both 8-HDFbinding pockets harbor similar nonpolar amino acids. However, in aCRY·8-HDF, F43 forms a π−π stack with 8-HDF providing a logical origin for the red shift, whereas the corresponding amino acid in (6−4) D. melanogaster is isoleucine 50. ...
... BiochemistryArticle DOI:10.1021/acs.biochem.9b00875Biochemistry 2020, 59, 594−604 in 8-HDF-binding photolyases such as the A. nidulans(6−4) photolyase, where a 25-fold decrease is observed,34,60 indicating a less efficient energy transfer between 8-HDF and FADH − in aCRY·8-HDF. ...
Cryptochromes are ubiquitous flavin-binding light sensors closely related to DNA repairing photolyases. The animal-like cryptochrome CraCRY from the green alga Chlamydomonas reinhardtii challenges the paradigm of cryptochromes as pure blue-light receptors by acting as a (6-4) photolyase, using 8-hydroxy-5-deazaflavin (8-HDF) as a light harvesting antenna with a 17.4 Å distance to flavin and showing spectral sensitivity up to 680 nm. The expanded action spectrum is attributed to the presence of the flavin neutral radical (FADH•) in the dark, despite a rapid FADH• decay observed in vitro in samples exclusively carrying flavin. Herein, the red-light response of CraCRY carrying flavin and 8-HDF was studied, revealing a 3-fold prolongation of the FADH• life-time in the presence of 8-HDF. Millisecond time-resolved UV-vis spectroscopy showed the red light-induced formation and decay of an absorbance band at 458 nm concomitant to flavin reduction. Time-resolved FTIR spectroscopy and density functional theory attributed these changes to the deprotonation of 8-HDF, challenging the paradigm of 8-HDF being permanently deprotonated in photolyases. FTIR spectra showed changes in the hydrogen bonding network of asparagine 395, a residue suggested to indirectly control the flavin protonation, indicating the involvement of N395 in the stabilization of FADH•. Fluorescence spectroscopy revealed a decrease in energy transfer efficiency of 8-HDF upon flavin reduction, possibly linked to the 8-HDF deprotonation. The discovery of the interdependence of flavin and 8-HDF beyond energy transfer processes highlights the essential role of the antenna, introducing a new concept enabling CraCRY and possibly other bifunctional cryptochromes to fulfill their dual function.
