The plant photoreceptor phototropin is an autophosphorylating serine-threonine protein kinase activated by UV-A/blue light. Two domains, LOV1 and LOV2, members of the PAS domain superfamily, mediate light sensing by phototropin. Heterologous expression studies have shown that both domains function as FMN-binding sites. Although three plant blue light photoreceptors, cry1, cry2, and phototropin, have been identified to date, the photochemical reactions underlying photoactivation of these light sensors have not been described so far. Herein, we demonstrate that the LOV domains of Avena sativa phototropin undergo a self-contained photocycle characterized by a loss of blue light absorbance in response to light and a spontaneous recovery of the blue light-absorbing form in the dark. Rate constants and quantum efficiencies for the photoreactions indicate that LOV1 exhibits a lower photosensitivity than LOV2. The spectral properties of the photoproduct produced for both LOV domains are unrelated to those found for photoreduced flavins and flavoproteins, but are consistent with those of a flavin-cysteinyl adduct. Flavin-thiol adducts are generally short-lifetime reaction intermediates formed during the flavoprotein-catalyzed reduction of protein disulfides. By site-directed mutagenesis, we have identified several amino acid residues within the putative chromophore binding site of LOV1 and LOV2 that appear to be important for FMN binding and/or the photochemical reactivity. Among those is Cys39, which plays an important role in the photochemical reaction of the LOV domains. Replacement of Cys39 with Ala abolished the photochemical reactions of both LOV domains. We therefore propose that light sensing by the phototropin LOV domains occurs via the formation of a stable adduct between the FMN chromophore and Cys39.

Phototropin is a membrane-bound UV-A/blue light photoreceptor of plants responsible for phototropism, chloroplast migration and stomatal opening. Characteristic are two LOV domains, each binding one flavin mononucleotide, in the Nterminal half and having a serine/threonine kinase domain in the C-terminal half of the molecule. We purified the N-terminal half of oat phototropin 1, containing LOV1 and LOV2 domains, as a soluble fusion protein with the calmodulin binding peptide (CBP) by expression in Escherichia coli. Gel chromatography showed that it was dimeric in solution. While the fusion protein CBP-LOV2 was exclusively monomeric in solution, the fusion protein CBP-LOV1 occurred as monomer and dimer. The proportion of dimer increased on prolonged incubation. We conclude that native phototropin is a dimer and that the LOV1 domain is probably responsible for dimerization.

Chlorophyll synthetase catalyzes the last step of chlorophyll biosynthesis, namely prenylation (esterification) of chlorophyllide with phytyl diphosphate or geranylgeranyl diphosphate. During investigation of various chlorophyllide derivatives as potential substrates we observed lower esterification with increasing percentages of chlorophyllide a' in epimeric mixtures of chlorophyllides a and a: To avoid epimerization during esterification, we studied the reaction in detail with model compounds [zinc-1 32(R)-methoxy-pheophorbide a and zinc-1 3'(S)-rnethoxy-pheophorbide a, zinc-1 32(R)-methoxy-pyropheophorbide a and zinc-chlorin e,-l3', 1 5'-dimethylester]. We conclude that compounds which have the 13'-carbomethoxy group at the same side of the macrocycle as the propionic side chain of ring D are neither substrates nor competitive inhibitors. Only compounds having the 132-carbomethoxy group at the opposite site are substrates for the enzyme. Naturally occuring chlorophyll a ' must be formed by epimerization after esterification.Chlorophyll a' [a-'prime', 13Z(S)-chlorophyll a ] has been known since 1942 (Strain and Manning, 1942) as a byproduct of isolation of chlorophyll a [13'(R)-chlorophyll a]. Due to the easy epimerization of chlorophyll at C-13' (Hynninen, 1991) it is generally believed that it is formed from chlorophyll a during the extraction procedure. However, increasing evidence has accumulated during the last decade that chlorophyll a' is a natural constituent of higher plants and cyanobacteria (Watanabe et al., 1985 a,b;Kobayashi et al., 1988). Investigations on pigment composition of Chlamydomonas reinhardtii (Maroc and Tremolieres, 1990) and of P700-enriched chloroplasts of higher plants (Maeda et al., 1992) revealed that two chlorophyll a' molecules are situated in the core of photosystem I. Furthermore, the presence of two bacteriochlorophyll g 'molecules in the reaction center of heliobacteria was also described (Kobayashi et al., 1990(Kobayashi et al., , 1991. The question now arises, at which stage of the biosynthetic pathway of the chlorophylls is the prime pigment synthesized, especially whether it is formed before or after esterification of chlorophyllide a.Chlorophyll synthetase catalyzes prenylation of chlorophyllides with geranylgeranyl diphosphate (GerGerP,) or phytyl diphosphate (PhyP,), the last step of chlorophyll biosynthesis (Rudiger et al., 1980). This step is essential for translation and accumulation of chlorophyll a apoproteins (Eichacker et al., 1990(Eichacker et al., , 1992 and probably for stable assembly also for other components of the thylakoid membrane (Paulsen et al., 1990; Rudiger 1992 Rudiger , 1993. Chlorophyll synthetase catalyzes prenylation not only of chlorophyllide a, but also of chlorophyllide b and some modified derivatives (Benz and Rudiger, 1981 and Rudiger, 1992;Vezitskii and Sherbakov, 1987). During our studies on the substrate specificity of chlorophyll synthetase, we observed fractions of chlorophyllide a with a greatly reduced ability for esterificat...

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