Formation and Function of Flavin Anion Radical in Cryptochrome 1 Blue-Light Photoreceptor of Monarch Butterfly

Song, Sang-Hun; Öztürk, Nuri; Denaro, Tracy R.; Arat, N. Özlem; Kao, Ya-Ting; Zhu, Haisun; Zhong, Dongping; Reppert, Steven M.; Sancar, Aziz

doi:10.1074/jbc.m702874200

Cited by 84 publications

(129 citation statements)

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“…17 The redox status of FAD photoreduction, the Trp triad and the effects of Cys mutations at the flavin binding site on the flavin photochemistry are listed in Table 1. To put these results in perspective we summarize our recent functional studies in which light-induced proteolysis of Type 1 CRYs in vivo was used as an endpoint for photoreceptor activity: 16,17 The C→N mutants showed the same photoinduced proteolysis as the wild type CRYs, excluding the possibility of photoadduction with the cysteine near the N5 position as the initial signaling steps, which has been observed in phototropin, 24,25 and suggests that FADHfunctions as well as FAD •-in insect Type 1 CRYs. In contrast, the C→A mutants in which flavin is readily photoreduced to FAD •-but reoxidizes within seconds (more than an order of magnitude faster than their wild-type counterparts) showed no photoreceptor activity in vivo , 17 indicating that a stable FAD •-but not oxidized FAD must be the active form of flavin in …”

Section: Steady-state Spectroscopic Properties Of Type 1 Crysmentioning

confidence: 99%

Ultrafast Dynamics and Anionic Active States of the Flavin Cofactor in Cryptochrome and Photolyase

et al. 2008

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We report here our systematic studies of the dynamics of four redox states of the flavin cofactor in both photolyases and insect Type 1 cryptochromes. With femtosecond resolution, we observed ultrafast photoreduction of oxidized state (FAD) in subpicosecond and of neutral radical semiquinone (FADH • ) in tens of picoseconds through intraprotein electron transfer mainly with a neighboring conserved tryptophan triad. Such ultrafast dynamics make these forms of flavin unlikely to be the functional states of the photolyase/cryptochrome family. In contrast, we find that upon excitation the anionic semiquinone (FAD •-) and hydroquinone (FADH -) have longer lifetimes that are compatible with high-efficiency intermolecular electron transfer reactions. In photolyases, the excited active state (FADH -* ) has a long (nanosecond) lifetime optimal for DNA-repair function. In insect Type 1 cryptochromes known to be blue-light photoreceptors the excited active form (FAD •-* ) has complex deactivation dynamics on the time scale from a few to hundreds of picoseconds, which is believed to occur through conical intersection(s) with a flexible bending motion to modulate the functional channel. These unique properties of anionic flavins suggest a universal mechanism of electron transfer for the initial functional steps of the photolyase/cryptochrome blue-light photoreceptor family.

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Section: Steady-state Spectroscopic Properties Of Type 1 Crysmentioning

confidence: 99%

Ultrafast Dynamics and Anionic Active States of the Flavin Cofactor in Cryptochrome and Photolyase

et al. 2008

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“…Because of the high sequence and structural similarities between photolyase and CRY, it is generally assumed that CRYs have the same two cofactors as well. However, no CRY has been purified to date from its native source and those that have been purified as recombinant proteins contain FAD to varying levels and either trace amounts of MTHF or none at all (Lin et al 1995;Malhotra et al 1995;Özgür and Sancar 2003;Song et al 2007). Hence, formal proof that CRYs contain MTHF, or any other secondary chromophore, is lacking.…”

Section: Structures Of Photolyase and Cryptochromementioning

confidence: 99%

“…This review is in large part based on papers by Sancar (2003, 2006), Sancar (2004), Partch (2006), and Song et al (2007).…”

Section: Acknowledgmentsmentioning

confidence: 99%

Structure and Function of Animal Cryptochromes

Öztürk

Song²,

Özgür³

et al. 2007

Cold Spring Harbor Symposia on Quantitative Biology

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Cryptochrome (CRY) is a photolyase-like flavoprotein with no DNA-repair activity but with known or presumed blue-light receptor function. Animal CRYs have DNA-binding and autokinase activities, and their flavin cofactor is reduced by photoinduced electron transfer. In Drosophila, CRY is a major circadian photoreceptor, and in mammals, the two CRY proteins are core components of the molecular clock and potential circadian photoreceptors. In mammals, CRYs participate in cell cycle regulation and the cellular response to DNA damage by controlling the expression of some cell cycle genes and by directly interacting with checkpoint proteins. in response to blue light (Koornneef et al. 1980), was isolated and sequenced, it revealed high sequence homology with Escherichia coli photolyase, and hence, it was in retrospect, correctly speculated that HY4 encoded a blue light photoreceptor and not a signal transducer involved in blue light response (Ahmad and Cashmore 1993). Later, this protein was named cryptochrome 1 (Lin et al. 1995, and a second Arabidopsis protein that has high homology with CRY1 identified by genomics was named CRY2 . The role of CRY in Arabidopsis growth (CRY1) and differentiation (CRY2) was well established by 1995 (see Guo et al. 1998). However, as late as 1997, it was thought that CRY had no role in circadian photoreception in plants (Millar and Kay 1997).The first report implicating CRY in the circadian clock of any organism, plant or animal, came about from the study of DNA repair, in particular, the repair of UV damage in humans by photolyase. This issue had been controversial for nearly 25 years when Li et al. (1993) conducted an exhaustive study with a highly specific and sensitive assay, concluding that humans, like all placental mammals, lacked photolyase (Li et al. 1993). However, a 1995 release of a human expressed sequence tag (EST) list contained a "photolyase ortholog" entry (Adams et al. 1995). In light of this finding and the discovery of a photolyase in Drosophila and rattlesnake (Todo et al. 1993Kim et al. 1996) that repairs the minor UV-induced lesion, the (6-4) photoproduct, in contrast to the classic photolyase that repairs cyclobutane pyrimidine dimers (CPDs), the earlier conclusion regarding the lack of photolyase in humans needed reevaluation. This was done by Hsu et al. (1996) who, in addition to the "photolyase ortholog" in public databases, discovered a second human photolyase gene. Human cells expressing both genes and recombinant proteins encoded by both genes were tested for CPD and (6-4) photolyase activities and were found to lack both. Moreover, the proteins encoded by these genes, like most photolyases (Johnson et al. 1988) and Arabidopsis CRY (Lin et al. 1995; Malhorta et al. 1995), contained FAD (flavin-adenine dinucleotide) and a pterin cofactor. Therefore, it was concluded that these proteins were not repair enzymes but that, like Arabidopsis CRYs, they performed non-repair-related blue light functions and were named human CRY1 and 2 (Hsu et al. 1996). In humans ...

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“…The active state of the cofactor in vivo is in the anionic hydroquinone form (FADH -) in photolyase (6), but currently the redox status of flavin in cryptochrome is under debate with some studies suggesting flavin to be in oxidized (FAD), whereas others claiming anionic (FAD -/FADH -) states for the functional form in vivo (7)(8)(9)(10). However, in vitro, the cofactor is oxidized to FAD and/or FADH • in photolyase but only appears in the oxidized FAD state in cryptochrome.…”

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confidence: 99%

Determining complete electron flow in the cofactor photoreduction of oxidized photolyase

Liu¹,

Tan

Guo³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The flavin cofactor in photoenzyme photolyase and photoreceptor cryptochrome may exist in an oxidized state and should be converted into reduced state(s) for biological functions. Such redox changes can be efficiently achieved by photoinduced electron transfer (ET) through a series of aromatic residues in the enzyme. Here, we report our complete characterization of photoreduction dynamics of photolyase with femtosecond resolution. With various site-directed mutations, we identified all possible electron donors in the enzyme and determined their ET timescales. The excited cofactor behaves as an electron sink to draw electron flow from a series of encircling aromatic molecules in three distinct layers from the active site in the center to the protein surface. The dominant electron flow follows the conserved tryptophan triad in a hopping pathway across the layers with multiple tunneling steps. These ET dynamics occur ultrafast in less than 150 ps and are strongly coupled with local protein and solvent relaxations. The reverse electron flow from the flavin is slow and in the nanosecond range to ensure high reduction efficiency. With 12 experimentally determined elementary ET steps and 6 ET reaction pairs, the enzyme exhibits a distinct reduction-potential gradient along the same aromatic residues with favorable reorganization energies to drive a highly unidirectional electron flow toward the active-site center from the protein surface.protein electron transfer | flavin photoreduction | femtosecond dynamics | electron flow directionality | reduction potential funnel P hotolyase and cryptochrome are evolutionally related and contain a flavin adenine dinucleotide (FAD) as the catalytic cofactor with a unique bent structure in the active sites, but the two perform different functions: photolyase repairs UV-damaged DNA and cryptochrome functions as a photoreceptor for regulation of plant growth or synchronization of circadian rhythm (1-5). The active state of the cofactor in vivo is in the anionic hydroquinone form (FADH -) in photolyase (6), but currently the redox status of flavin in cryptochrome is under debate with some studies suggesting flavin to be in oxidized (FAD), whereas others claiming anionic (FAD -/FADH -) states for the functional form in vivo (7-10). However, in vitro, the cofactor is oxidized to FAD and/or FADH • in photolyase but only appears in the oxidized FAD state in cryptochrome. Thus, it appears that reduction of the oxidized FAD is necessary for its transformation into the active state. Photoinduced electron transfer (ET) is an effective way for such redox state changes and it is conceivable that the photoreduction of FAD could be a primary process for signal initiation in cryptochrome (11).In this study, we used Escherichia coli photolyase (EcPL) as a model system for systematic studies of electron flow into the excited cofactor FAD. As shown in Fig. 1, more than 10 aromatic acid residues (W and Y) encircle the flavin cofactor around the active site (12). These aromatic residues not only are cri...

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Formation and Function of Flavin Anion Radical in Cryptochrome 1 Blue-Light Photoreceptor of Monarch Butterfly

Cited by 84 publications

References 27 publications

Ultrafast Dynamics and Anionic Active States of the Flavin Cofactor in Cryptochrome and Photolyase

Ultrafast Dynamics and Anionic Active States of the Flavin Cofactor in Cryptochrome and Photolyase

Structure and Function of Animal Cryptochromes

Determining complete electron flow in the cofactor photoreduction of oxidized photolyase

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