Photoreaction Cycle of the Light, Oxygen, and Voltage Domain in FKF1 Determined by Low-Temperature Absorption Spectroscopy ² Kazunori Zikihara, ‡,§ Tatsuya Iwata, | Daisuke Matsuoka, ‡ Hideki Kandori, | Takeshi Todo, § and Satoru Tokutomi* ,‡ Department of Biological Science, Graduate School of Science, Osaka Prefecture UniVersity, 1-1 Gakuencho, Sakai, Osaka 599-8531, Japan, Department of Material Science and Engineering, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan, and Radiation Biology Center, Graduate School of Medicine, Kyoto UniVersity, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan ReceiVed April 22, 2006; ReVised Manuscript ReceiVed July 3, 2006 ABSTRACT: Flavin-binding Kelch repeat F-box (FKF1) protein plays important roles in the photoregulation of flowering in Arabidopsis. FKF1 has a light, oxygen, and voltage (LOV) sensing domain binding a flavin mononucleotide (FMN) as a chromophore noncovalently. Photoreaction of the FKF1-LOV polypeptide was studied by low-temperature absorption spectroscopy. Upon blue light irradiation, a ground state, D 450 , is converted to S 390 known as a cysteinyl-flavin adduct intermediate in the photoreaction of phototropin. Below 150 K, bleaching of D 450 was much reduced and a new photoproduct, Z 370 , appeared as well as S 390 formation. The calculated absorption spectrum for Z 370 is very similar to those of flavoproteins in an anion radical state. On the basis of the results that S 390 formation proceeds to Z 370 formation and that Z 370 formed at low temperatures reverts to D 450 upon temperature increase, Z 370 is concluded to be not an intermediate from D 450 to S 390 .Z 370 is suggested to be formed from the biradical triplet-excited state after relaxing to the ground state with the FMN anion radical trapped at the low temperature, in which the SH of the cysteine is in the wrong position that is able to produce a radical pair but unable to form the cysteinyl-flavin adduct. The counter SH in the cationic radical state may revert to the ground state by extracting an electron from the unidentified amino acid residue. Interestingly, S 390 that has been thought to be irreversible to D 450 was revealed to revert to D 450 very slowly with a half-life time of 62.5 h in solution at 298 K. The photoreaction mechanism is discussed in reference to the calculated activation energy of the reaction processes. Plants have acquired three major photoreceptive pigments, a red/far-red photoreversible receptor, phytochrome, and two blue light receptors, cryptochrome and phototropin (phot), 1 to sense their environmental conditions during the evolutional processes. Phot (1, 2) was first identified as a photoreceptor for tropic responses in Arabidopsis thaliana (A. thaliana) (3) and then proved to be a photoreceptor for chloroplast relocation (4, 5) and stomata opening (6). Phot has two chromophoric domains in the N-terminal half named LOV (light, oxygen, and voltage) (7-9) that bind a FMN nonco- valently. The LOV domains are known to be a subfamily of the PER-ARNT-SIM (PAS) superfamily thought to act as protein-protein interaction modules in eukaryotic cellular signaling (10). Phot has isoforms phot1 and phot2, in A. thaliana, that show different light sensitivity and share the regulatory functions (11). In addition to the phot families, A. thaliana has three more unique proteins that have only one LOV domain, named ZTL (LKP1, ADO1) (12-14), LKP2 (ADO2) (15, 16), and FKF1 (LKP3, ADO3) (17). All of them have three characteristic domains, a LOV domain, a F-box, and a Kelch repeat in this order from the N- to the C-termini. The function of the F-box is to interact with SKP proteins that are a component of the SCF (Skp/Cullin/F-box) class of E3 ubiquitin ligases that link the target substrates to the core ubiquitinating activity of the ligase complex (18, 19). Kelch repeat, while, is known to act as a protein-protein interacting site (16, 20). FKF1 has been shown to be involved in the light- regulated expression of CONSTANS (CO)(17), one of a key gene in day-length discrimination leading to flowering under long-day conditions through controlling the expression of a gene, Flowering Locus T (FT)(21-23), by enhancing degradation of CDF1, a repressor of CO expression (24) possibly through an ubiquitin-proteasome protein degrada- tion pathway (25-27). The binding site between FKF1 and CDF1 is reported to be in the Kelch repeat region (24), and ² This work was supported by grants-in-aid from MEXT Japan (Nos. 13139205 and 17084008 to S.T. and No. 14658215 to H.K.) and by the NITECH 21th Century COE Program to T.I. * To whom correspondence should be addressed. Phone: +81-72-254-9841. Fax: +81-72-254-9841. E-mail: toxan@ riast.osakafu-u.ac.jp. ‡ Osaka Prefecture University. § Kyoto University. | Nagoya Institute of Technology. 1 Abbreviations: A. thaliana, Arabidopsis thaliana; CO, CONSTANS; E. coli, Escherichia coli; FKF, flavin-binding Kelch repeat F-box; FMN, flavin mononucleotide; FT, Flowering Locus T; GST, glutathione S-transferase; LKP, LOV Kelch protein; LOV, light, oxygen, and voltage; NMR, nuclear magnetic resonance; PAGE, polyacrylamide gel electrophoresis; PAS, PER-ARNT-SIM; phot, phototropin; phot1, phototropin 1; phot2, phototropin 2; phy3, phytochrome 3; ZTL, ZEITLUPE. 10828 Biochemistry 2006, 45, 10828-10837 10.1021/bi0607857 CCC: $33.50 © 2006 American Chemical Society Published on Web 08/16/2006