Phytochromes are widely distributed biliprotein photoreceptors. Typically, the chromophore becomes covalently linked to the protein during an autocatalytic lyase reaction. Plant and cyanobacterial phytochromes incorporate bilins with a ring A ethylidene side chain, whereas other bacterial phytochromes utilize biliverdin as chromophore, which has a vinyl ring A side chain. For Agrobacterium phytochrome Agp1, site-directed mutagenesis provided evidence that biliverdin is bound to cysteine 20. This cysteine is highly conserved within bacterial homologues, but its role as attachment site has as yet not been proven. We therefore performed mass spectrometry studies on proteolytic holopeptide fragments. For that purpose, an Agp1 expression vector was re-engineered to produce a protein with an N-terminal affinity tag. Following proteolysis, the chromophore co-purified with a ca. 5 kDa fragment during affinity chromatography, showing that the attachment site is located close to the N-terminus. Mass spectrometry analyses performed with the purified chromopeptide confirmed the role of the cysteine 20 as biliverdin attachment site. We also analyzed the role of the highly conserved histidine 250 by site-directed mutagenesis. The homologous amino acid plays an important but yet undefined role in plant phytochromes and has been proposed as chromophore attachment site of Deinococcus phytochrome. We found that in Agp1, this amino acid is dispensable for covalent attachment, but required for tight chromophore-protein interaction.
The kinetics and mechanism of the autocatalytic assembly of holo-Cph1 phytochrome (from Synechocystis) from the apoprotein and the bilin chromophores phycocyanobilin (PCB) and phycoerythrobilin (PEB) were investigated by stopped flow and circular dichroism. At 1:1 stoichiometry, pH 7.9, and 10 degrees C, SVD analysis of the kinetic data for PCB revealed three spectral components involving three transitions with time constants tau(1) approximately 150 ms, tau(2) approximately 2.5 s, and tau(3) approximately 50 s. Tau(1) was associated with a major red shift and transfer of oscillator strength from the Soret region to the 680 nm region. When the sulfhydryl group of cysteine 259 was blocked with iodoacetamide, preventing the formation of a covalent adduct, a noncovalent red-shifted complex (680 nm) was formed with a time constant of 200 ms. Tau(1) could thus be assigned to the formation of a noncovalent complex. The absorption changes during tau(1) are due to the formation of the extended conformation of the linear tetrapyrrole and to its protonation in the binding pocket. From the concentration and pH dependence of the kinetics we obtained a value of 1.5 microM for the K(D) of this noncovalent complex and a value of 8.4 for the pK(a) of the proton donor. The tau(2) component was associated with a blue shift of about 25 nm and was attributed to the formation of the covalent bond (P(r)), accompanied with the loss of the 3-3' double bond to ring A. Tau(3) was due to photoconversion to P(fr). For PEB, which is not photochromic, the formation of the noncovalent complex is faster (tau(1) = 70 ms), but the covalent bond formation is about 80 times slower (tau(2) = 200 s) than with the natural chromophore PCB. The CD spectra of the PCB adduct in the 250-800 nm range show that the chromophore geometries in P(r) and P(fr) are similar to those in plant phytochrome. The opposite rotational strengths of P(r) and P(fr) in the longest wavelength band suggest that the photoisomerization induces a reversal of the chirality. The Cph1 complex with noncovalently bound PCB was still photochromic when cysteine 259 was blocked with IAA or with the bulkier IAF. The covalent linkage to cysteine 259 is thus not required for photoconversion. The CD spectra of the noncovalently bound PCB in P(r)- and P(fr)-like states are qualitatively similar to those of the covalent adducts, suggesting analogous structures in the binding pocket. The noncovalent interactions with the binding pocket are apparently sufficient to hold the chromophore in the appropriate geometry for photoisomerization.
Phytochromes are biliprotein photoreceptors that are found in plants, bacteria, and fungi. Prototypical phytochromes have a Pr ground state that absorbs in the red spectral range and is converted by light into the Pfr form, which absorbs longer-wavelength, far-red light. Recently, some bacterial phytochromes have been described that undergo dark conversion of Pr to Pfr and thus have a Pfr ground state. We show here that such so-called bathy phytochromes are widely distributed among bacteria that belong to the order Rhizobiales. We measured in vivo spectral properties and the direction of dark conversion for species which have either one or two phytochrome genes. Agrobacterium tumefaciens C58 contains one bathy phytochrome and a second phytochrome which undergoes dark conversion of Pfr to Pr in vivo. The related species Agrobacterium vitis S4 contains also one bathy phytochrome and another phytochrome with novel spectral properties. Rhizobium leguminosarum 3841, Rhizobium etli CIAT652, and Azorhizobium caulinodans ORS571 contain a single phytochrome of the bathy type, whereas Xanthobacter autotrophicus Py2 contains a single phytochrome with dark conversion of Pfr to Pr. We propose that bathy phytochromes are adaptations to the light regime in the soil. Most bacterial phytochromes are light-regulated histidine kinases, some of which have a C-terminal response regulator subunit on the same protein. According to our phylogenetic studies, the group of phytochromes with this domain arrangement has evolved from a bathy phytochrome progenitor.
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