Active site of DNA photolyase: tryptophan-306 is the intrinsic hydrogen atom donor essential for flavin radical photoreduction and DNA repair in vitro

Li, Ywan Feng; Heelis, Paul F.; Sancar, Aziz

doi:10.1021/bi00239a034

Cited by 159 publications

(185 citation statements)

References 39 publications

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“…We stress that the identity of RP2 in AtCry is not crucial for the interpretation of the magnetic-field effects reported below. In light of previous work (9,10,24,28,29), the Trp radicals are presumed to come from the terminal residue of the Trp-triad, i.e. Trp-324 in AtCry and Trp-306 in EcPL.…”

Section: Resultsmentioning

confidence: 99%

Magnetically sensitive light-induced reactions in cryptochrome are consistent with its proposed role as a magnetoreceptor

Maeda

Robinson

Henbest

et al. 2012

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

309

480

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Among the biological phenomena that fall within the emerging field of “quantum biology” is the suggestion that magnetically sensitive chemical reactions are responsible for the magnetic compass of migratory birds. It has been proposed that transient radical pairs are formed by photo-induced electron transfer reactions in cryptochrome proteins and that their coherent spin dynamics are influenced by the geomagnetic field leading to changes in the quantum yield of the signaling state of the protein. Despite a variety of supporting evidence, it is still not clear whether cryptochromes have the properties required to respond to magnetic interactions orders of magnitude weaker than the thermal energy, k B T . Here we demonstrate that the kinetics and quantum yields of photo-induced flavin—tryptophan radical pairs in cryptochrome are indeed magnetically sensitive. The mechanistic origin of the magnetic field effect is clarified, its dependence on the strength of the magnetic field measured, and the rates of relevant spin-dependent, spin-independent, and spin-decoherence processes determined. We argue that cryptochrome is fit for purpose as a chemical magnetoreceptor.

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Section: Resultsmentioning

confidence: 99%

Magnetically sensitive light-induced reactions in cryptochrome are consistent with its proposed role as a magnetoreceptor

Maeda

Robinson

Henbest

et al. 2012

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

309

480

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“…In E. coli photolyase, the "Trp triad," FADH°←Trp-382←Trp-359←Trp-306 is the predominant photoreduction pathway with Trp-306 being the ultimate electron donor (Fig. 8A) (Li et al 1991;Park et al 1995;Kavakli and Sancar 2004). Interestingly, the residues of this "Trp triad" are conserved in most DNA photolyases and CRYs Lin and Shalitin 2003;Zeugner et al 2005).…”

Section: The "Trp Triad" and Photoreductionmentioning

confidence: 99%

“…Interestingly, the residues of this "Trp triad" are conserved in most DNA photolyases and CRYs Lin and Shalitin 2003;Zeugner et al 2005). It has been unequivocally shown that this pathway has no role in the photocycle of photolyase (Li et al 1991;Kavaklı and Sancar 2004). In contrast, as will be discussed below, some studies have suggested that photoinduced electron 126 ÖZTÜRK ET AL.…”

Section: The "Trp Triad" and Photoreductionmentioning

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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“…1). Earlier biochemical studies (14) as well as X-ray structures (12) identified the conserved tryptophan triad (W382, W359, and W306) as the main electron source for photoreduction, and the recent ultrafast studies (15)(16)(17) of semiquinone FADH • photoreduction revealed the continuing hopping mechanism along the tryptophan triad. The hopping mechanism, consisting of multiple tunneling steps, has been shown to significantly accelerate electron transport in DNA (18) and also recently in proteins (19).…”

mentioning

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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170

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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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Active site of DNA photolyase: tryptophan-306 is the intrinsic hydrogen atom donor essential for flavin radical photoreduction and DNA repair in vitro

Cited by 159 publications

References 39 publications

Magnetically sensitive light-induced reactions in cryptochrome are consistent with its proposed role as a magnetoreceptor

Magnetically sensitive light-induced reactions in cryptochrome are consistent with its proposed role as a magnetoreceptor

Structure and Function of Animal Cryptochromes

Determining complete electron flow in the cofactor photoreduction of oxidized photolyase

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