Spectral and photochemical diversity of tandem cysteine cyanobacterial phytochromes

Abstract

The atypical trichromatic cyanobacterial phytochrome NpTP1 from Nostoc punctiforme ATCC 29133 is a linear tetrapyrrole (bilin)-binding photoreceptor protein that possesses tandem-cysteine residues responsible for shifting its light-sensing maximum to the violet spectral region. Using bioinformatics and phylogenetic analyses, here we established that tandem-cysteine cyanobacterial phytochromes (TCCPs) compose a well-supported monophyletic phytochrome lineage distinct from prototypical red/far-red cyanobacterial phytochromes. To investigate the light-sensing diversity of this family, we compared the spectroscopic properties of NpTP1 (here renamed NpTCCP) with those of three phylogenetically diverged TCCPs identified in the draft genomes of Tolypothrix sp. PCC7910, Scytonema sp. PCC10023, and Gloeocapsa sp. PCC7513. Recombinant photosensory core modules of ToTCCP, ScTCCP, and GlTCCP exhibited violet-blue-absorbing dark-states consistent with dual thioether-linked phycocyanobilin (PCB) chromophores. Photoexcitation generated singly-linked photoproduct mixtures with variable ratios of yellow-orange and red-absorbing species. The photoproduct ratio was strongly influenced by pH and by mutagenesis of TCCP- and phytochrome-specific signature residues. Our experiments support the conclusion that both photoproduct species possess protonated 15E bilin chromophores, but differ in the ionization state of the noncanonical "second" cysteine sulfhydryl group. We found that the ionization state of this and other residues influences subsequent conformational change and downstream signal transmission. We also show that tandem-cysteine phytochromes present in eukaryotes possess similar amino acid substitutions within their chromophore-binding pocket, which tune their spectral properties in an analogous fashion. Taken together, our findings provide a roadmap for tailoring the wavelength specificity of plant phytochromes to optimize plant performance in diverse natural and artificial light environments.

Keywords: Color sensing; algae; cyanobacteria; dual cysteine phytochromes; photoreceptor; photosynthesis; phycocyanobilin; plant; protochromism; violet and blue light photoreceptors.

Figure 1.

Proposed TCCP photocycle and SyCph1 GAF domain structure labeled with chromophore-containing signature residues. A, proposed photocycle. TCCP dark-states absorb violet-to-blue light due to two thioether linkages to the PCB chromophore (15). Light absorption by the ^15ZPvb “dark-state” triggers ultrafast isomerization of the 15Z double bond of the doubly-linked PCB chromophore that destabilizes the thioether linkage at C10 to generate a YO-absorbing ^15EPyo “ion pair” photoproduct with a re-aromatized chromophore. The ^15EPyo photoproduct equilibrates with a R-absorbing ^15EPr photoproduct, a process requiring Asp-205 and favored at low pH (as shown by this investigation). We envisage this process to entail a repositioning of the D-ring to interact with the Asp-205 carboxylate, likely in conjunction with rearrangement of the tongue region of the adjacent PHY domain seen in other phytochromes (30, 31, 48). This interconversion can be very fast (<seconds) or quite slow (>minute, as seen for NpTCCP) depending on TCCP species or variant. The ^15EPyo/^15EPr equilibrium also varies among TCCP family members and can be altered by site-directed mutations of conserved TCCP-signature residues in both GAF and PHY domains (as shown by this investigation). Both ^15EPyo and ^15EPr photoproduct states are photoactive because YO or R light can trigger regeneration of the ^15EPvb dark-state. Residues in ToTCCP, which likely interact with the chromophore are shown in parentheses. B, chromophore-contacting signature residues of SyCph1 and corresponding residues found in ToTCCP. Shown is the chromophore-binding pocket of the SyCph1 GAF domain with PCB in the R-absorbing Pr state (PDB ID 2VEA) (28). The chromophore-binding pocket is drawn in surface representation (light gray). Labels refer to amino acid sequence numbers, and PCB is colored in teal and shown in stick form. Key chromophore-contacting residues are labeled with black font for SyCph1. ToTCCP residues conserved with or diverged from those in Cph1 are labeled (in brackets) with red or blue fonts, respectively

Figure 2.

Phylogenetic analysis of photosensory core modules of TCCP, Cph1/CphA, and BphP/CphB family representatives supports a monophyletic TCCP lineage in cyanobacteria. The TCCP family consisting of NpTCCP (NpF1183) from N. punctiforme ATCC 29133 (15), orthologues ToTCCP from Tolypothrix PCC7910 (Tpr0787), ScTCCP from Scytonema PCC10023 (Spr6432), and GlTCCP from Gloeocapsa PCC7513 (Gpr2095) studied here (indicated with arrows), comprise a monophyletic clade distinct from Cph1/CphA and BphP/CphB cyanobacterial phytochrome families. Noncyanobacterial BphPs are included as an outgroup. Detailed sequence names, accessions, and sequence alignments are included as in Data File S1.

Figure 3.

Spectral properties of bilin adducts of PCMs of recombinant ToTCCP, ScTCCP, and GlTCCP proteins indicate differences in the ratio of two spectrally distinct 15E photoproducts in the TCCP family. Absorption spectra of PCMs of ToTCCP (A), ScTCCP (B), and GlTCCP (C) incorporating PCB (solid line), BV (dotted line), or PΦB (dashed line) are shown in 15Z dark-states (blue) and 15E photoproducts (orange) in standard assay buffer at pH 8 (see “Experimental procedures”). Dark-minus-light difference spectra at pH 8 for native ToTCCP (D), ScTCCP (E), and GlTCCP (F) were obtained from panels A–C, respectively. Zinc in-gel fluorescence and Coomassie Brilliant Blue (CBB) images of acid-denatured TCCPs (G) are shown. SDS-PAGE gels imaged by zinc-dependent in-gel fluorescence (upper panel) and after staining with CBB (lower panel) are shown. Molecular mass markers (M) in kDa are indicated on the ordinate on the right side.

Figure 4.

Absorption spectra of Cys variants of ToTCCP indicates an essential role for the TCCP-signature cysteine in forming the B-absorbing 15Z dark-state. Absorption spectra of PCMs of ToTCCP, canonical Cys variants, C257A (A) and C257S (B), and TCCP-signature cysteine variants, C258H (C), C258S (D), C258D (E), C258I (F), C258F (G), and C258Y (H), at pH 8 incorporating PCB are shown for 15Z dark-states (blue) and 15E photoproducts (orange). The loss of the canonical Cys in C257A/C257S variants yields a B-absorbing 15Z species that affords a stable B-absorbing 15E state because of the apparent stability of the C10 linkage. The loss of the TCCP-signature Cys (C258H/C258S/C258D/C258I/C258F/C258Y) yields red-absorbing variants in both 15Z and 15E states due to the loss of the C10 thioether linkage.

Figure 5.

Absorption spectra of ToTCCP variants in TCCP family-specific, ToTCCP-specific, and TCCP-family variable residues reveal selective roles in spectral tuning in the TCCP family. 15Z dark-states are shown in blue, 15E photoproduct states shown in orange before and after dark maturation (solid and dashed, respectively). Zinc in-gel fluorescence data for all mutants are shown in Fig. S3.

Figure 6.

Spectroscopic pH titrations of ToTCCP and its singly-linked C258S variant reveals pH-dependent equilibria between G- and R-absorbing species. ToTCCP WT and C258S variant in 15Z dark and 15E lit states at pH 8 were adjusted to different pH values (see “Experimental procedures” for details). All spectra are normalized to the same concentration in panels A, B, D, and E. In panels C and F, the 15E photoproduct at pH 7 and 10 (solid curves, blue pH 10, and orange pH 7) were adjusted to pH 7 or 10 (dashed curves, blue pH 7 and orange pH 10). Normalized spectra shown are superimposed.

Figure 7.

pH dependence of selected variants of ToTCCP indicates that the 15E photoproduct comprises a mixture of spectrally distinct YO-absorbing “deprotonated” and R-absorbing “protonated” states. ToTCCP variants after conversion to their 15E photoproduct states were adjusted to different pH values (see “Experimental procedures” for details). Spectra for each indicated variant are colored by pH as follows: pH 6 (red), pH 8 (green), and pH 11 (fushia).