The Photocycle of a Flavin-binding Domain of the Blue Light Photoreceptor Phototropin

Abstract

The plant blue light receptor, phot1, a member of the phototropin family (1), is a plasma membrane-associated flavoprotein that contains two (ϳ110 amino acids) flavinbinding domains, LOV1 and LOV2, within its N terminus and a typical serine-threonine protein kinase domain at its C terminus. The LOV (light, oxygen, and voltage) domains belong to the PAS domain superfamily of sensor proteins. In response to blue light, phototropins undergo autophosphorylation. E. coli-expressed LOV domains bind riboflavin-5-monophosphate, are photochemically active, and have major absorption peaks at 360 and 450 nm, with the 450 nm peak having vibronic structure at 425 and 475 nm. These spectral features correspond to the action spectrum for phototropism in higher plants. Near-UV blue light regulates a variety of different responses in higher plants. These include phototropism, the inhibition of hypocotyl elongation, the expression of various genes, and stomatal opening. Phot1 (nph1), the recently discovered blue light receptor, is a member of the phototropin receptor family (1). Phot1 is a plasma membrane-associated flavoprotein that functions as the primary photoreceptor mediating phototropic plant movement (2-4). Phot1 has two 12.1-kDa flavin-binding domains, LOV1 and LOV2, within its N-terminal region and a typical serinethreonine protein kinase domain at the C-terminal region. Heterologous expression studies have shown that phot1 binds FMN 1 as a chromophore and undergoes autophosphorylation in response to light treatment. It has therefore been proposed that this receptor functions as a light-activated serine/threonine kinase (4). The isolated LOV domains from oat phot1 expressed in Escherichia coli have been shown to undergo a cyclic photoreaction upon the absorption of light; LOV1 recovers with a half-time of 11.5 s, whereas LOV2 recovers with a half-time of 27 s (5). In addition, the quantum efficiencies for photoproduct (adduct) formation for LOV1 and LOV2 are ϳ0.045 and 0.44, respectively (5). The ground forms of the LOV domains have major absorption peaks at 360 and 450 nm with the 450 peak having vibronic structure at 425 and 475 nm. Upon absorption of light, the chromophore bleaches 2 in the 450 nm region generating a species that absorbs maximally at 390 nm. This intermediate has been assigned as a flavin-cysteinyl adduct between the protein and the C(4a) carbon of the FMN chromophore. This adduct breaks down spontaneously, returning the protein to its ground form. A LOV2 mutant (LOV2C39A) in which the cysteine that forms the adduct has been mutated to alanine does not undergo this photoreaction (5).Recently the crystal structure of the LOV2 domain from the fern Adiantum capillus-veneris phy3 (6) was solved to 2.7-Å resolution (7). Phy3 is a chimeric photoreceptor with homology to phytochrome at its N-terminal end and an almost complete phototropin at its C-terminal end. Its LOV2 domain shares a 70% sequence homology to the oat phot1 LOV2 (6). The structure indicates that the FMN molecule is held noncovalently within...

The LOV2 Domain of Phototropin: A Reversible Photochromic Switch

“…Φ F in LOV2 has been estimated between 0.3 and 0. 44,5,7,10 implying that Φ B ranges between 0.2 and 0.3, in good agreement with the estimate from our ultrafast experiments.In conclusion, we find that, upon absorption of near-UV light by the LOV2 S 390 state, the covalent bond between the flavin and the conserved cysteine is broken and the blue-light-sensitive groundstate D 447 is regenerated on an ultrafast time scale of 100 ps. Thus, LOV2 is a reversible photochromic switch, which can be "turned on" by blue/near-UV light, and "turned off" by near-UV light, as schematically shown in Scheme 1.…”

“…Φ F in LOV2 has been estimated between 0.3 and 0. 44,5,7,10 implying that Φ B ranges between 0.2 and 0.3, in good agreement with the estimate from our ultrafast experiments.…”

“…3,4 Blue-light absorption initiates a photochemical reaction which results in the formation of a covalent adduct between a conserved cysteine and the flavin. 5,6 It is believed that this species, referred to as S 390 given its absorption band in the near-UV, corresponds to the signaling state of the protein. The lifetime of the adduct in various LOV domains ranges from minutes to hours, 5,7-9 which implies that even under physiological illumination, there is a high probability for absorption of a second, near-UV photon.…”

The Role of Key Amino Acids in the Photoactivation Pathway of the Synechocystis Slr1694 BLUF Domain

“…To estimate the FAD T SADS, the ground state bleach part from 420 to 500 nm was set identical to that of the singlet excited state FAD* and a low weight was assigned to the FAD T SADS between 420 and 500 nm assigned in the fitting procedure to avoid compensation effects with flavin ground state bleach present in the SADS of FAD* 1-4 , Q 1 , Q 2 , and Slr RED . The resulting FAD T SADS shows a broad, positive absorption from 500 to 690 nm, with a spectral shape that is similar to that observed in the AppA BLUF domain (35) and the LOV 1 and LOV 2 domains of phototropin (46,52,53).…”

Effect of computational methodology on the conformational dynamics of the protein photosensor LOV1 from Chlamydomonas reinhardtii

“…In this context, it is worth pointing out that in LOV domains an efficient ISC rate is crucial. This relates to the fact that the biologically active adduct state is generated from the first excited triplet state of FMN, which is itself formed through ISC from its first excited singlet state with a high quantum yield of about 60-80% [11,70,71,[73][74][75]. In several experimental studies [12,70,71], it has been speculated that the enhanced ISC rate in LOV domains is caused by a heavy-atom effect induced by an interaction between the cysteine sulfur and the flavin isoalloxazine ring.…”