Green/red cyanobacteriochromes regulate complementary chromatic acclimation via a protochromic photocycle

Abstract

Cyanobacteriochromes (CBCRs) are cyanobacterial members of the phytochrome superfamily of photosensors. Like phytochromes, CBCRs convert between two photostates by photoisomerization of a covalently bound linear tetrapyrrole (bilin) chromophore. Although phytochromes are red/far-red sensors, CBCRs exhibit diverse photocycles spanning the visible spectrum and the near-UV (330-680 nm). Two CBCR subfamilies detect near-UV to blue light (330-450 nm) via a "two-Cys photocycle" that couples bilin 15Z/15E photoisomerization with formation or elimination of a second bilin-cysteine adduct. On the other hand, mechanisms for tuning the absorption between the green and red regions of the spectrum have not been elucidated as of yet. CcaS and RcaE are members of a CBCR subfamily that regulates complementary chromatic acclimation, in which cyanobacteria optimize light-harvesting antennae in response to green or red ambient light. CcaS has been shown to undergo a green/red photocycle: reversible photoconversion between a green-absorbing 15Z state ((15Z)P(g)) and a red-absorbing 15E state ((15E)P(r)). We demonstrate that RcaE from Fremyella diplosiphon undergoes the same photocycle and exhibits light-regulated kinase activity. In both RcaE and CcaS, the bilin chromophore is deprotonated as (15Z)P(g) but protonated as (15E)P(r). This change of bilin protonation state is modulated by three key residues that are conserved in green/red CBCRs. We therefore designate the photocycle of green/red CBCRs a "protochromic photocycle," in which the dramatic change from green to red absorption is not induced by initial bilin photoisomerization but by a subsequent change in bilin protonation state.

Fig. 1.

RcaE from F. diplosiphon is a light-regulated protein kinase. (A, Upper) Cells of F. diplosiphon that were fully acclimated to green light (GL) or red light (RL). (Lower) Domain architecture of full-length RcaE (705 residues). (B) Absorption and (C) CD spectra of the ^15ZP_g (green lines) and ^15EP_r (red lines) forms of the truncated GAF domain from RcaE at pH 7.5. (B, Insets) Colors of the ^15ZP_g and ^15EP_r forms of the GAF domain in solution. (D) Autophosphorylation of full-length RcaE in its ^15ZP_g and ^15EP_r forms. Incorporation of ³²P was quantified with a PhosphorImager. (Inset) Original autoradiograph.

Fig. 2.

Protochromic absorption changes of RcaE. (A) Absorbance spectra of the RcaE 15Z state are shown for pH values between 5.0 (red) and 11.0 (blue) in 0.5 pH unit increments. The composite inset shows the colors of the RcaE solutions between pH 5.5 (left) and 11.0 (right). (B) Absorbance spectra and a composite inset are shown for the 15E state of RcaE at the same pH values as in A. (C) Absorbance values at the indicated wavelengths were plotted vs. pH for the RcaE 15Z state. Data were fit to a model having one titrating group (Eq. S6). (D) A similar analysis was performed for the RcaE 15E state, using a model having two titrating groups (Eq. S9). Data for 593 nm (gray) were magnified 15× for clarity; the axis runs from 0.1 to 0.12. (E) RcaE is shown after in vitro reconstitution with PCB or 15Za-PCB. Proteins were analyzed by SDS/PAGE followed by staining with Coomassie Brilliant Blue (CBB, Upper) or zinc blotting (Lower). The asterisk indicates a contaminating protein found in apoprotein preparations and derived from E. coli cells. (F) Photocycle of holo-RcaE reconstituted with 15Za-PCB (black) or PCB (green and red) in vitro. No change in absorption was observed after prolonged irradiation with green light for 15Za-PCB–reconstituted RcaE.

Fig. 3.

Identification of a protochromic triad regulating the bilin pK_a. (A) RcaE proteins were analyzed by SDS/PAGE, followed by CBB staining (Upper) or zinc blotting (Lower). (B) Absorption spectra are shown for Y₁₈₇F, L₂₄₉H, E₂₁₇Q, E₂₁₇D, and K₂₆₁M RcaE proteins in the 15Z state (green) and 15E state (red) at pH 7.5. Absorption spectra are shown for wild-type RcaE at pH 10.0 in the same color scheme for comparison. The percentage of total holoprotein converted to the 15Z and 15E states was estimated by acid denaturation followed by absorption spectroscopy and is indicated for each state. (C) pH titrations of E₂₁₇A and K₂₆₁M RcaE were performed as described for wild-type RcaE in Fig. 2.

Fig. 4.

A mechanism for the protochromic photocycle. Bilin photoisomerization does not cause the spectral shift between green and red absorption, but instead modulates the bilin pK_a to trigger proton transfer to or from Glu217. Protonated ^15ZP_r and deprotonated ^15EP_g intermediates identified in our pH titration study and the protochromic triad identified by site-directed mutagenesis are shown. P, propionate.