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Animal Type 1 Cryptochromes

2007; Elsevier BV; Volume: 283; Issue: 6 Linguagem: Inglês

10.1074/jbc.m708612200

1083-351X

Nuri Öztürk, Sang-Hun Song, Christopher P. Selby, Aziz Sancar,

Photoreceptor and optogenetics research

It has recently been realized that animal cryptochromes (CRYs) fall into two broad groups. Type 1 CRYs, the prototype of which is the Drosophila CRY, that is known to be a circadian photoreceptor. Type 2 CRYs, the prototypes of which are human CRY 1 and CRY 2, are known to function as core clock proteins. The mechanism of photosignaling by the Type 1 CRYs is not well understood. We recently reported that the flavin cofactor of the Type 1 CRY of the monarch butterfly may be in the form of flavin anion radical, FAD·¯, in vivo. Here we describe the purification and characterization of wild-type and mutant forms of Type 1 CRYs from fruit fly, butterfly, mosquito, and silk moth. Cryptochromes from all four sources contain FADox when purified, and the flavin is readily reduced to FAD·¯ by light. Interestingly, mutations that block photoreduction in vitro do not affect the photoreceptor activities of these CRYs, but mutations that reduce the stability of FAD·¯ in vitro abolish the photoreceptor function of Type 1 CRYs in vivo. Collectively, our data provide strong evidence for functional similarities of Type 1 CRYs across insect species and further support the proposal that FAD·¯ represents the ground state and not the excited state of the flavin cofactor in Type 1 CRYs. It has recently been realized that animal cryptochromes (CRYs) fall into two broad groups. Type 1 CRYs, the prototype of which is the Drosophila CRY, that is known to be a circadian photoreceptor. Type 2 CRYs, the prototypes of which are human CRY 1 and CRY 2, are known to function as core clock proteins. The mechanism of photosignaling by the Type 1 CRYs is not well understood. We recently reported that the flavin cofactor of the Type 1 CRY of the monarch butterfly may be in the form of flavin anion radical, FAD·¯, in vivo. Here we describe the purification and characterization of wild-type and mutant forms of Type 1 CRYs from fruit fly, butterfly, mosquito, and silk moth. Cryptochromes from all four sources contain FADox when purified, and the flavin is readily reduced to FAD·¯ by light. Interestingly, mutations that block photoreduction in vitro do not affect the photoreceptor activities of these CRYs, but mutations that reduce the stability of FAD·¯ in vitro abolish the photoreceptor function of Type 1 CRYs in vivo. Collectively, our data provide strong evidence for functional similarities of Type 1 CRYs across insect species and further support the proposal that FAD·¯ represents the ground state and not the excited state of the flavin cofactor in Type 1 CRYs. Cryptochromes are photolyase-related flavoproteins that play important roles in regulating the circadian clock in animals and growth and development in plants (1Lin C. Shalitin D. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2003; 54: 469-496Crossref PubMed Scopus (380) Google Scholar, 2Cashmore A.R. Cell. 2003; 114: 537-543Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 3Sancar A. J. Biol. Chem. 2004; 279: 34079-34082Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The mechanism of photosignaling by animal cryptochromes is not known. Previously, it was thought that CRYs 3The abbreviations used are: S2 cellsSchneider 2 cellsCRYcryptochromeTim proteinTimeless protein. 3The abbreviations used are: S2 cellsSchneider 2 cellsCRYcryptochromeTim proteinTimeless protein. in Drosophila and other insects function as circadian photoreceptors and in mouse and other vertebrates function as core components of the molecular clock (4Partch C.L. Sancar A. Photochem. Photobiol. 2005; 81: 1291-1304Crossref PubMed Scopus (105) Google Scholar). Recently, this view was revised when it was realized that some insects such as the honeybee possess only a mammalian CRY-like cryptochrome and others such as the monarch butterfly possess both Drosophila CRY-like and mammalian CRY-like cryptochromes (5Zhu H. Yuan Q. Briscoe A.D. Froy O. Casselman A. Reppert S.M. Curr. Biol. 2005; 15: 953-954Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 6Yuan Q. Metterville D. Briscoe A.D. Reppert S.M. Mol. Biol. Evol. 2007; 24: 948-955Crossref PubMed Scopus (254) Google Scholar). It was proposed that Drosophila-like CRYs should be referred to as Type 1 CRYs and the mammalian-like CRYs should be referred to as Type 2 CRYs (6Yuan Q. Metterville D. Briscoe A.D. Reppert S.M. Mol. Biol. Evol. 2007; 24: 948-955Crossref PubMed Scopus (254) Google Scholar). Furthermore, it was found that all Type 1 CRYs tested were subject to light-induced proteolysis in Schneider 2 (S2) cells and, hence, were considered to function as circadian photoreceptors in a manner analogous to DmCRY (6Yuan Q. Metterville D. Briscoe A.D. Reppert S.M. Mol. Biol. Evol. 2007; 24: 948-955Crossref PubMed Scopus (254) Google Scholar). Similarly, it was shown that insect Type 2 CRYs, like the mammalian CRYs, functioned as core clock proteins with no demonstrable photoreceptor activity (6Yuan Q. Metterville D. Briscoe A.D. Reppert S.M. Mol. Biol. Evol. 2007; 24: 948-955Crossref PubMed Scopus (254) Google Scholar). Schneider 2 cells cryptochrome Timeless protein. Schneider 2 cells cryptochrome Timeless protein. We are interested in the photoreceptor function of CRY and specifically in the cryptochrome photocycle. Type 1 CRYs are well suited for this purpose because their photoinitiated proteolysis constitutes a convenient functional assay (7Emery P. So W.V. Kaneko M. Hall J.C. Rosbash M. Cell. 1998; 95: 669-679Abstract Full Text Full Text PDF PubMed Scopus (701) Google Scholar, 8Stanewsky R. Kaneko M. Emery P. Beretta B. Wager-Smith K. Kay S.A. Rosbash M. Hall J.C. Cell. 1988; 95: 681-692Abstract Full Text Full Text PDF Scopus (773) Google Scholar, 9Van Vickle-Chavez S.J. Van Gelder R.N. J. Biol. Chem. 2007; 282: 10561-10566Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Two recent studies reported that Type 1 CRYs from Drosophila melanogaster and the monarch butterfly (Danaus plexippus), purified as recombinant proteins, contained near-stoichiometric amounts of flavin in the two-electron oxidized, FADox, form. Exposure of these CRYs to light reduced the flavin to the flavin anion semiquinone, FAD·¯, with high quantum yield (10Song S-H. Öztűrk N. Denaro T.R. Arat N.Ö. Kao Y.T. Zhu H. Zhong D. Reppert S.M. Sancar A. J. Biol. Chem. 2007; 282: 17608-17612Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 11Berndt A. Kottke T. Breitkreuz H. Dvorsky R. Henning S. Alexander M. Wolf E. J. Biol. Chem. 2007; 282: 13011-13021Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Whereas it was speculated that this photoreduction reaction constituted the initial step of the DmCRY photocycle (11Berndt A. Kottke T. Breitkreuz H. Dvorsky R. Henning S. Alexander M. Wolf E. J. Biol. Chem. 2007; 282: 13011-13021Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), experiments with DpCRY1 did not support this model as a general feature of Type 1 CRYs (10Song S-H. Öztűrk N. Denaro T.R. Arat N.Ö. Kao Y.T. Zhu H. Zhong D. Reppert S.M. Sancar A. J. Biol. Chem. 2007; 282: 17608-17612Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar); it was found that a non-photoreducible mutant of DpCRY1 exhibited photoinduced proteolysis kinetics in S2 cells identical to that of wild-type DpCRY1 and, hence, it was proposed that FAD·¯ actually represented the ground state of flavin in DpCRY1 and by extension in all Type 1 CRYs in vivo. It was, therefore, proposed that FAD·¯ was converted to FADox during protein purification under aerobic conditions (10Song S-H. Öztűrk N. Denaro T.R. Arat N.Ö. Kao Y.T. Zhu H. Zhong D. Reppert S.M. Sancar A. J. Biol. Chem. 2007; 282: 17608-17612Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). In this study we extend the previous investigations by purifying wild-type and mutant Type 1 CRYs from four species: D. melanogaster, D. plexippus (monarch butterfly), Anopheles gambiae (mosquito), and Antheraea pernyi (Chinese oak silk moth). We find remarkable similarities among the Type 1 CRYs from these species with respect to their spectroscopic and photochemical and photobiological properties. Significantly, we extend the observation that blocking the in vitro photoreduction pathway does not affect the photoreceptor function in vivo and quite unexpectedly we find that Type 1 CRY mutants which cannot accommodate FAD·¯ but possess FADH· flavin neutral radical are equally sensitive to photoinduced proteolysis as the wild-type photoreceptors. Cloning of Type 1 CRYs into Bacterial and Insect Cell Expression VectorsThe cDNAs of Type 1 CRYs from D. plexippus, A. gambiae, and A. pernyi (6Yuan Q. Metterville D. Briscoe A.D. Reppert S.M. Mol. Biol. Evol. 2007; 24: 948-955Crossref PubMed Scopus (254) Google Scholar) were cloned into the pMal-c2 bacterial expression vector (New England Biolabs) by standard methods. The cloned genes were sequenced to ensure there were no accidental mutations. The resulting constructs expressed Type 1 CRYs as fusion proteins attached to the C-terminal end of Escherichia coli maltose-binding protein. Site-directed mutations in the cloned genes were introduced by standard methods using the QuikChange method (Stratagene). A viral vector for expressing D. melanogaster CRY with FLAG and His tags at the N terminus was prepared using the Invitrogen Bac-to-Bac Baculovirus expression system. The cDNAs of Type 1 CRYs were also inserted into the pAc5.1v5/HisA vector for transient transfection into S2 cells to investigate photoinduced CRY degradation. Site-directed mutations of the subcloned genes were made by standard methods and verified by sequencing. In addition, the cDNA of DpCRY2 (negative control) and the β-galactosidase gene (loading control) were inserted into the same vector for co-transfection along with the Type 1 CRY clones (5Zhu H. Yuan Q. Briscoe A.D. Froy O. Casselman A. Reppert S.M. Curr. Biol. 2005; 15: 953-954Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Purification of Type 1 CRYsThe maltose-binding protein-CRY fusion proteins were expressed in E. coli BL21 strain (Stratagene) and purified as described previously for DpCRY1 (10Song S-H. Öztűrk N. Denaro T.R. Arat N.Ö. Kao Y.T. Zhu H. Zhong D. Reppert S.M. Sancar A. J. Biol. Chem. 2007; 282: 17608-17612Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Typical yields were about 2 mg of CRY from a 12-liter culture. The purified proteins contained essentially stoichiometric amount of FAD. They were not analyzed for the presence of the folate cofactor, which we previously reported to be present in trace amounts in DpCRY1 preparations (10Song S-H. Öztűrk N. Denaro T.R. Arat N.Ö. Kao Y.T. Zhu H. Zhong D. Reppert S.M. Sancar A. J. Biol. Chem. 2007; 282: 17608-17612Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The purified proteins were kept at -80 °C in storage buffer containing 50 mm Tris-HCl, pH 7.5, 100 mm KCl, 5 mm dithiothreitol, and 50% (v/v) glycerol. Purification and handling of CRYs were carried out under dim yellow light (λ > 550 nm) to prevent accidental photoreduction. To purify DmCRY, Sf9 cells were infected with the DmCRY baculovirus, and the cells were harvested 2 days later. Cells were lysed as described previously (12Özgür S. Sancar A. Biochemistry. 2006; 45: 13369-13374Crossref PubMed Scopus (46) Google Scholar), and the protein was purified using anti-FLAG M2-agarose resin from Sigma. Both wild-type and mutant proteins were obtained at ∼0.3 mg/300-ml culture and contained near-stoichiometric flavin. PhotoreductionCryptochromes in storage buffer were irradiated with 450 nm (10-nm bandwidth) in a monochromator (150-watt xenon lamp, Photon Technology International). The fluence rate was 11.8 ergs·mm-2·s-1. Absorption spectra were recorded using a Shimadzu UV-601 spectrophotometer. Some photoreduction experiments were carried out with 366 nm from black light (General Electric) as described previously (10Song S-H. Öztűrk N. Denaro T.R. Arat N.Ö. Kao Y.T. Zhu H. Zhong D. Reppert S.M. Sancar A. J. Biol. Chem. 2007; 282: 17608-17612Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Photoinduced Type 1 CRY DegradationS2 cells transfected with appropriate plasmids were exposed to 366-nm light at a rate of 1 milliwatt·cm-2, and the levels of the target CRYs and control proteins were determined by Western blotting with monoclonal anti-V5 IgG (Invitrogen) as described previously (10Song S-H. Öztűrk N. Denaro T.R. Arat N.Ö. Kao Y.T. Zhu H. Zhong D. Reppert S.M. Sancar A. J. Biol. Chem. 2007; 282: 17608-17612Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Mutant DmCRY (W342F) was poorly expressed and typically 5-fold more vector of this mutant was needed than the vector expressing the wild-type protein to obtain comparable levels of expression. The levels of CRYs were expressed relative to the β-galactosidase internal control and quantified using ImageQuant 5.0 software (GE Healthcare). Purification of Insect Type 1 CryptochromesPreviously, we reported the purification of monarch butterfly CRY1 (DpCRY1) expressed in E. coli with the maltose-binding protein tag (10Song S-H. Öztűrk N. Denaro T.R. Arat N.Ö. Kao Y.T. Zhu H. Zhong D. Reppert S.M. Sancar A. J. Biol. Chem. 2007; 282: 17608-17612Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). This tag aids both in solubilization and purification of the fused proteins. Hence, we expressed Type 1 CRYs from A. gambiae (AgCRY1) and A. pernyi (ApCRY1) as maltose-binding protein fusion proteins as well and purified them by affinity chromatography on amylose resin. Our previous attempt to purify D. melanogaster CRY (DmCRY) in this manner yielded an enzyme preparation with grossly substoichiometric flavin content (13Selby C.P. Sancar A. Photochem. Photobiol. 1999; 69: 105-107Crossref PubMed Scopus (28) Google Scholar). A recent report indicated that DmCRY expressed in a baculovirus/Sf21 vector/host system contains high levels of flavin (11Berndt A. Kottke T. Breitkreuz H. Dvorsky R. Henning S. Alexander M. Wolf E. J. Biol. Chem. 2007; 282: 13011-13021Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Independently, we expressed FLAG-tagged DmCRY in Sf9 cells and purified DmCRY with nearly stoichiometric content of FAD. Fig. 1 shows the absorption spectra of Type 1 CRYs of these four insect species. The absorption spectra look remarkably similar and exhibit vibrational fine structures characteristic of protein-bound flavin. Importantly, in all enzymes FADox is converted to FAD·¯ by light exposure (Fig. 1) indicating that the similarities among these enzymes extends beyond the phylogenetic relationship and is reflected in their spectroscopic/photochemical properties as well. Photochemical Properties of Type 1 CRYs with Mutations in the "Trp Triad" and the Flavin Binding SiteFlavin photoreduction in photolyase, plant, and animal CRYs is thought to proceed in large part by electron transport through three Trp residues that correspond to Trp-306 (distal)%Trp-359 %Trp-382 (proximal) residues in E. coli photolyase (14Sancar A. Chem. Rev. 2003; 103: 2203-2238Crossref PubMed Scopus (1026) Google Scholar, 15Li Y.F. Heelis P.F. Sancar A. Biochemistry. 1991; 30: 6322-6329Crossref PubMed Scopus (151) Google Scholar, 16Park H.W. Kim S.T. Sancar A. Deisenhofer J. Science. 1995; 268: 1866-1872Crossref PubMed Scopus (497) Google Scholar, 17Kao Y.T. Saxena C. Wang L. Sancar A. Zhong D. Cell Biochem. Biophys. 2007; 48: 32-44Crossref PubMed Scopus (37) Google Scholar) in which the flavin photoreduction through the Trp triad was discovered (Fig. 2A). The photoreduction reaction generates flavin neutral radical in Arabidopsis thaliana CRYs (18Lin. C. Ahmad M. Gordon D. Cashmore A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8423-8427Crossref PubMed Scopus (163) Google Scholar, 19Zeugner A. Byrdin M. Bouly J.P. Bakrim N. Giovani B. Brettel K. Ahmad M. J. Biol. Chem. 2005; 280: 19437-19440Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 20Bouly J.P. Schleicher E. Dionisio-Sese M. Vandenbussche F. Van Der Stradeten D. Bakrim N. Meier S. Batschauer A. Galland P. Bittl R. Ahmad, M. Ahmad, M. J. Biol. Chem. 2007; 282: 9383-9391Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 21Banerjee R. Schleicher E. Meier S. Viana R.M. Pokorny R. Ahmad M. Bittl R. Batschauer A. J. Biol. Chem. 2007; 282: 14916-14922Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar) and flavin anion radical in DpCRY1 and DmCRY (10Song S-H. Öztűrk N. Denaro T.R. Arat N.Ö. Kao Y.T. Zhu H. Zhong D. Reppert S.M. Sancar A. J. Biol. Chem. 2007; 282: 17608-17612Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 11Berndt A. Kottke T. Breitkreuz H. Dvorsky R. Henning S. Alexander M. Wolf E. J. Biol. Chem. 2007; 282: 13011-13021Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). It was proposed (11Berndt A. Kottke T. Breitkreuz H. Dvorsky R. Henning S. Alexander M. Wolf E. J. Biol. Chem. 2007; 282: 13011-13021Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar) that the Asp-396 of AtCRY1 opposite N5 of the isoalloxazine ring acts as a proton donor during photoreduction of FADox through the Trp triad and, thus, generates FADH· by proton-coupled electron transfer (Fig. 2B). At the corresponding position, Type 1 CRYs contain a Cys residue. It was suggested that because Cys has a pKa more than four units higher than that of Asp, it could not act as a proton donor during photoreduction, thus explaining the generation of FAD·¯ and not FADH· in Type 1 CRYs (11Berndt A. Kottke T. Breitkreuz H. Dvorsky R. Henning S. Alexander M. Wolf E. J. Biol. Chem. 2007; 282: 13011-13021Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). To test these models for the pathways of electron and proton transfer during photoreduction, we performed site-directed mutagenesis in Type 1 CRYs to either block the proposed electron transport path or open the proposed proton transport route. It should be noted that the mutants of all Type 1 CRYs were not equally expressed or equally soluble. Hence, for our analysis we chose those enzymes that tolerated a given mutation the best as evidenced by their level of overproduction, solubility, and absorption spectra. The Trp Triad and PhotoreductionWe previously reported that the DpCRY1-W328F mutant was not photoreducible, supporting the role of the Trp triad in Type 1 CRY photoreduction (10Song S-H. Öztűrk N. Denaro T.R. Arat N.Ö. Kao Y.T. Zhu H. Zhong D. Reppert S.M. Sancar A. J. Biol. Chem. 2007; 282: 17608-17612Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). To generalize this observation we made the corresponding mutation in DmCRY (DmCRY-W342F) as well as the DpCRY1-W406F (proximal electron donor) and tested them for photoreduction. The results are shown in Fig. 3A. The replacement of Trp with redox inactive Phe in either the proximal (DpCRY1-Trp-406) or ultimate (DpCRY1-Trp-328 and DmCRY-Trp 342) electron donor blocked photoreduction, supporting the model of electron transfer through the Trp triad. It must be noted, however, that high doses of irradiation photoreduced 5–10% of DmCRY-W342F, suggesting a minor alternative pathway of photoreduction (data not shown). When the Trp residue corresponding to the middle Trp of the triad (W359 in E. coli photolyase) was changed to Phe, the mutant proteins of all Type 1 CRYs tested lacked flavin, presumably because of misfolding, and hence, the contribution of this Trp to electron transport during photoreduction could not be tested. With this caveat, we believe our data strongly support the notion that the Trp triad is the major route for photoreduction in Type 1 CRYs. Flavin Binding Site and PhotoreductionPhotoreduction of FADox in AtCRY1 and AtCRY2 first generates the FADH· neutral radical, which is quite stable (20Bouly J.P. Schleicher E. Dionisio-Sese M. Vandenbussche F. Van Der Stradeten D. Bakrim N. Meier S. Batschauer A. Galland P. Bittl R. Ahmad, M. Ahmad, M. J. Biol. Chem. 2007; 282: 9383-9391Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 21Banerjee R. Schleicher E. Meier S. Viana R.M. Pokorny R. Ahmad M. Bittl R. Batschauer A. J. Biol. Chem. 2007; 282: 14916-14922Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Further illumination produces the two-electron reduced and presumably deprotonated flavin, FADH- (20Bouly J.P. Schleicher E. Dionisio-Sese M. Vandenbussche F. Van Der Stradeten D. Bakrim N. Meier S. Batschauer A. Galland P. Bittl R. Ahmad, M. Ahmad, M. J. Biol. Chem. 2007; 282: 9383-9391Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). The latter is rather unstable under aerobic conditions and rapidly re-oxidizes to the FADH· and more slowly to the FADox form. It was rather surprising, therefore, to find that photoreduction of Type 1 CRYs generates the one-electron reduced and deprotonated flavin, FAD·¯. It has been suggested that the reason for this difference between plant CRYs and animal Type 1 CRYs is that in plant CRYs an Asp is located opposite the N5 of the isoalloxazine ring, whereas in Type 1 CRYs there is a Cys in that position, and Asp can donate a proton to reduced flavin because of its low pKa (∼3.9), whereas Cys cannot because of its much higher pKa (∼8.3) (11Berndt A. Kottke T. Breitkreuz H. Dvorsky R. Henning S. Alexander M. Wolf E. J. Biol. Chem. 2007; 282: 13011-13021Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). To test this model we replaced the Cys residue with Asp, Ala, or Asn in AgCRY1 and with Ala or Asn in ApCRY1 and analyzed the mutants for photoreduction. To our surprise the FAD in AgCRY1 with the Cys → Asp replacement was reduced to FAD·¯ and not to FADH· as predicted by the model (Fig. 3B). This finding suggests that the Asp in the flavin binding pocket is not the proton donor in the proton-coupled electron transfer reaction during photoreduction of FADox to FADH·. In fact, even the Cys → Ala mutants of both AgCRY1 and ApCRY1 were photoreduced to FAD·¯, suggesting that so far as the amino acid residue opposite N5 of the isoalloxazine ring is concerned there is no difference among the three amino acids (Cys, Asp, and Ala) with vastly different proton donating potentials. In contrast and quite unexpectedly, replacement of Cys by Asn led to formation of FADH· by photoreduction, consistent with electron transfer through the Trp triad coupled with a proton transfer from an amino acid residue at the flavin binding pocket. However, this amino acid is unlikely to be the Asn because the pKa of Asn (∼17) is nearly twice the pKa of Cys (∼8.3), which cannot act as proton donor. It is noteworthy that at the corresponding position E. coli photolyase contains Asn (16Park H.W. Kim S.T. Sancar A. Deisenhofer J. Science. 1995; 268: 1866-1872Crossref PubMed Scopus (497) Google Scholar). However, in E. coli photolyase the flavin is in the FADH· form before photoreduction, and photoreduction further reduces the flavin to the FADH· form (14Sancar A. Chem. Rev. 2003; 103: 2203-2238Crossref PubMed Scopus (1026) Google Scholar). In light of these findings we conclude that the reduced flavin takes up a proton not from an amino acid but from an acidic water molecule in the water network within the close confines of the active site of the photolyase/cryptochrome family. Quantitative Analysis of Photoreduction and ReoxidationNext we analyzed the photoreduction of wild-type and mutant CRYs as a function of light dose to obtain the quantum yields for the photoreactions as well as the rates of oxidation of reduced CRYs so as to be able to correlate the flavin reduction/oxidation thermodynamics and kinetics with the biological responses of the mutant proteins. The quantum yield of photoreduction is obtained from the Rupert plot (22Rupert C.S. J. Gen. Physiol. 1962; 45: 725-741Crossref PubMed Scopus (61) Google Scholar, 23Payne G. Sancar A. Biochemistry. 1990; 29: 7715-7727Crossref PubMed Scopus (103) Google Scholar) in which the fraction of remaining substrate, in this case FADox, is plotted as a function of the light dose on a semi-log plot (Rupert plot),ln([FADox]t/[FADox]0)=-kpL(Eq. 1) where L is the light dose in erg·mm-2, and ko is the photolytic constant, which is related to the quantum yield of the photoreaction by ϵϕ (m-1cm-1) = 5.2 x 109· kp (mm2 erg-1)·λ-1 (nm), where ϵ = molar extinction coefficient, ϕ = quantum yield, and λ = the wavelength of irradiation. Rupert plots for photoreduction of the Trp triad mutants are shown in Fig. 4, A and B, and the plots for photoreduction of the flavin binding site mutants are shown in Fig. 4, C and D. The photolytic cross section and quantum yield values are listed in Table 1. The following conclusions can be made from these data. First, all of the Type 1 CRYs tested are photoreduced with quantum yields of ϕ = 0.16–0.18. These are considerably higher than the quantum yields of photoreduction of photolyase (14Sancar A. Chem. Rev. 2003; 103: 2203-2238Crossref PubMed Scopus (1026) Google Scholar) and AtCRYs (20Bouly J.P. Schleicher E. Dionisio-Sese M. Vandenbussche F. Van Der Stradeten D. Bakrim N. Meier S. Batschauer A. Galland P. Bittl R. Ahmad, M. Ahmad, M. J. Biol. Chem. 2007; 282: 9383-9391Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 21Banerjee R. Schleicher E. Meier S. Viana R.M. Pokorny R. Ahmad M. Bittl R. Batschauer A. J. Biol. Chem. 2007; 282: 14916-14922Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Second, the Trp triad mutations with non-redox active amino acids essentially eliminate photoreduction, although at high light doses some residual photoreduction is observed in DmCRY-W342F (data not shown) as has been observed with E. coli photolyase W306F mutant (14Sancar A. Chem. Rev. 2003; 103: 2203-2238Crossref PubMed Scopus (1026) Google Scholar, 15Li Y.F. Heelis P.F. Sancar A. Biochemistry. 1991; 30: 6322-6329Crossref PubMed Scopus (151) Google Scholar). Third, mutation of the Cys residue in the flavin binding pocket affects the photoreduction quantum yield in an interesting pattern; Cys → Asp mutation does not significantly affect ϕ, Cys → Ala mutation reduces the quantum yields by about a factor of 2, and the Cys → Asn mutation increases the quantum yield by approximately a factor of 2. However, it must be noted that photoreduction of the Cys → Asn mutants generates the flavin neutral radical, FADH·, and not the FAD·¯ observed in the wild-type, the Cys → Asp, and the Cys → Ala mutants. Conceivably, H-bonding between the N5 of the isoalloxazine ring and the NH2 group of Asn increases the quantum yield by stabilizing the neutral radical and reducing the rate of back electron transfer.TABLE 1Photoreduction of FADox and reoxidation in select members of the Type1 CRYs and their mutants The quantum yield is calculated based on the extinction coefficient of 1.1 × 104 m–1·cm–1 at 450 nm for FADox. τ = life time of the reduced form under aerobic conditions. There was no measurable photoreduction of DmCRY-W342F, DpCRY1-W328F, and DpCRY1-W406F under our irradiation conditions.ProteinPhotoreductionReoxidation, τkpϕminDmCry-wild type1.52 × 10–40.1594.5DmCry-W342FDpCry1-wild type1.74 × 10–40.1830.91DpCry1-W328FDpCry1-W406FApCry1-wild type1.73 × 10–40.1821.8ApCry1-C402A9.78 × 10–50.1020.21ApCry1-C402N3.92 × 10–40.411FAD·¯, 1.1 FADH·, overnightAgCry1-wild type1.66 × 10–40.1744AgCry1-C413D1.54 × 10–40.16222AgCry1-C413A8.80 × 10–50.0930.25AgCry1-C413N3.97 × 10–40.417FAD·¯, 1.3 FADH·, overnight Open table in a new tab Next we determined the rates of reoxidation of wild-type and mutant CRYs that had been photoreduced (Fig. 5). Interestingly, the wild-type proteins differed in their reoxidation rates by as much as a factor of 5, with DpCRY1 having the fastest (life time (τ) = 0.9 min) and DmCRY having the slowest (τ = 4.5 min) reoxidation rates (Table 1). Of equal significance, mutations of the Cys residue in the flavin binding pocket have drastic effects on the rates of reoxidation (Table 1); in AgCRY1 FAD·¯ has a lifetime of 4 min, which increases to 22 min in the C413D mutant and decreases by 16-fold to 0.25 min in the C413A mutant. The latter value may explain the apparent low quantum yield of photoreduction of this mutant as during photoreduction there is significant reoxidation on the time scale of the photoreduction treatment. Most strikingly, the lifetime of FADH· in both AgCRY1-C413N and ApCRY1-C402N is ∼12 h under our assay conditions, although a small fraction of the flavin that is in the form of FAD·¯ after photoreduction in these mutants reoxidizes with rates comparable with those of the wild-type enzymes. Photoreceptor Functions of Type 1 CRYs with Mutations in the Trp Triad and the Flavin Binding SiteHaving analyzed some of the factors that affect the photoreduction of and the stability of the photoreduced flavin, we then proceeded to measure the effect of these factors on the photoreceptor function of cryptochromes. Currently, there are two assays for the photoreceptor functions of Type 1 CRYs; that is, photoinduced degradation of CRY itself and photoinduced degradation of the Timeless (Tim) protein (8Stanewsky R. Kaneko M. Emery P. Beretta B. Wager-Smith K. Kay S.A. Rosbash M. Hall J.C. Cell. 1988; 95: 681-692Abstract Full Text Full Text PDF Scopus (773) Google Scholar, 24Busza A. Emery-Le M. Rosbash M. Emery P. Science. 2004; 304: 1503-1506Crossref PubMed Scopus (234) Google Scholar). Although Tim proteolysis, by virtue of the photochemical reaction, is CRY-dependent, the photoinduced degradation of CRY is independent of Tim (24Busza A. Emery-Le M. Rosbash M. Emery P. Science. 2004; 304: 1503-1506Crossre