An optomechanical transducer in the blue light receptor phototropin from Avena sativa

Abstract

The PHOT1 (NPH1) gene from Avena sativa specifies the blue light receptor for phototropism, phototropin, which comprises two FMN-binding LOV domains and a serine͞threonine protein kinase domain. Light exposure is conducive to autophosphorylation of the protein kinase domain. We have reconstituted a recombinant LOV2 domain of A. sativa phototropin with various 13 C͞ 15 N-labeled isotopomers of the cofactor, FMN. The reconstituted protein samples were analyzed by NMR spectroscopy under dark and light conditions. Blue light irradiation is shown to result in the addition of a thiol group (cysteine 450) to the 4a position of the FMN chromophore. The adduct reverts spontaneously in the dark by elimination. The light-driven flavin adduct formation results in conformational modification, which was diagnosed by 1 H and 31 P NMR spectroscopy. This conformational change is proposed to initiate the transmission of the light signal via conformational modulation of the protein kinase domain conducive to autophosphorylation of NPH1. N umerous phenomena in the life cycle of plants, including phototropism, stomatal opening, and circadian periodicity, are modulated by light. Photoreceptors responsive to UV, blue, red, and far red light have been reported. Together, they span the spectral range of about 280-800 nm.Blue light-responsive processes have been known for a long time, but cognate receptors have been characterized only recently. Thus, cryptochromes characterized by sequence similarity to DNA photolyases are now believed to be involved in the synchronization of the circadian clock (1-3). Phototropins are involved in phototropism (4, 5) and have no sequence similarity with the cryptochrome group. FAD and FMN serve as chromophores for cryptochromes and phototropins, respectively (6).Phototropin of Avena sativa is a protein with 923 amino acids specified by the NPH1 gene (ref. 7; for review, see also ref. 8).The protein comprises two FMN-binding LOV domains and a serine͞threonine protein kinase domain.The LOV domains of phototropin are similar to PAS domains involved in light, oxygen, or voltage sensing (4) in a variety of regulatory as well as sensor proteins exhibiting diverse functions (4, 9, 10). Recombinant LOV1 and LOV2 domains of phototropins from different plants have been shown to bind FMN (11). The crystal structure of the LOV2 domain of PHY3 protein of Adiantum capillus-veneris has been published recently (12).In this study, we reconstituted the LOV2 apoprotein from A. sativa with Experimental ProceduresMaterials. Stable isotope-labeled FMN samples were prepared by published procedures (13-15). Recombinant LOV2 Domain of Phototropin.A recombinant fusion protein comprising the calmodulin-binding domain from myosin light chain kinase and the LOV2 domain from NPH1 protein of A. sativa was prepared as described earlier (16,17). The fusion protein is subsequently designated recombinant LOV2 domain.Reconstitution of LOV2 Domain with Isotope-Labeled FMN. Recombinant LOV2 domain (fusion protein) was depleted of FMN and su...

Blue Light Perception in Plants

“…Given that adduct formation proceeds via 3 FMN, then FMNH ϩ and the adduct must also be created in the triplet state. That the adduct would be created in a triplet state is rather unlikely, but if it were, its spin state would have been evident from its effect on the NMR line widths, yet no line broadening was observed (22). The only possibility is that FMNH ϩ converts to the singlet ground state before the adduct is formed.…”

“…Hence, in the wild type there must be a competition between adduct formation and the electron transfer reaction with another as yet unidentified amino acid residue. Nevertheless, adduct formation is the overwhelmingly dominant process in wild-type LOV2; no radicals are observed in the wild type that may be irradiated (reversibly) for long periods without suffering degradation (22). Previous optical studies have determined that adduct formation occurs on the microsecond time scale (20,48).…”

“…Until now, however, the details of the mechanism of adduct formation have not been conclusively established. The ground state, an intermediate state resembling the FMN triplet ( 3 FMN), and the adduct (19,20) have been observed in phototropin by optical spectroscopy, whereas with 13 C and 31 P NMR (22) and x-ray crystallography (24) the ground state and the adduct could be characterized. Swartz et al (20) propose an ionic reaction pathway for FMN-C(4a)-thiol adduct formation with the Cys-450 residue initially present as a thiolate, thus requiring a proton donor other than Cys-450 to protonate the light-generated FMN triplet (20).…”

COS1: An Arabidopsis coronatine insensitive1 Suppressor Essential for Regulation of Jasmonate-Mediated Plant Defense and Senescence

“…Lumazine synthase catalyzes the penultimate step in biosynthesis of riboflavin (vitamin B2) in plants, fungi, and microorganisms (Persson et al, 1999), which is a key component in the riboflavin pathway essential for diverse yet critical cellular processes. Riboflavin is essential for the basic metabolism and serves as a precursor of coenzymes riboflavin monophosphate and flavin adenine dinucleotide; the riboflavin pathway-derived flavocoenzymes were found to function as indispensable redox cofactors in all cells and also serve a variety of other roles, such as DNA photorepair, light sensing, and bioluminescence (Muller, 1992;Sancar, 1994;Ahmad et al, 1998;Christie et al, 1998;Vande Berg and Sancar, 1998;Briggs and Huala, 1999;Aubert et al, 2000;Briggs and Olney, 2001;Salomon et al, 2001; Q.Y. .…”

Structure of 3,4-Dihydroxy-2-butanone 4-Phosphate Synthase from Methanococcus jannaschii in Complex with Divalent Metal Ions and the Substrate Ribulose 5-Phosphate

“…Its derivatives, flavin mononucleotide (FMN) and flavinadenine dinucleotide (FAD), are indispensable in all cells where they serve a variety of redox reactions (1). They have also been shown to serve a variety of other functions in cells such as DNA photorepair (2), light sensing (3,4), and bioluminescence (5)(6)(7)(8) and in reactions without net-redox change (9).…”