Protochromic absorption changes in the two-cysteine photocycle of a blue/orange cyanobacteriochrome

Abstract

Edited by Ruma Banerjee Cyanobacteriochromes (CBCRs) are phytochrome-related photosensors with diverse spectral sensitivities spanning the entire visible spectrum. They covalently bind bilin chromophores via conserved cysteine residues and undergo 15Z/15E bilin photoisomerization upon light illumination. CBCR subfamilies absorbing violet-blue light use an additional cysteine residue to form a second bilin-thiol adduct in a two-Cys photocycle. However, the process of second thiol adduct formation is incompletely understood, especially the involvement of the bilin protonation state. Here, we focused on the Oscil6304_2705 protein from the cyanobacterium Oscillatoria acuminata PCC 6304, which photoconverts between a blue-absorbing 15Z state (15Z Pb) and orange-absorbing 15E state (15E Po). pH titration analysis revealed that 15Z Pb was stable over a wide pH range, suggesting that bilin protonation is stabilized by a second thiol adduct. As revealed by resonance Raman spectroscopy, 15E Po harbored protonated bilin at both acidic and neutral pH, but readily converted to a deprotonated green-absorbing 15Z state (15Z Pg) at alkaline pH. Site-directed mutagenesis revealed that the conserved Asp-71 and His-102 residues are required for second thiol adduct formation in 15Z Pb and bilin protonation in 15E Po, respectively. An Oscil6304_2705 variant lacking the second cysteine residue, Cys-73, photoconverted between deprotonated 15Z Pg and protonated 15E Pr, similarly to the protochromic photocycle of the green/red CBCR subfamily. Timeresolved spectroscopy revealed 15Z Pg formation as an intermediate in the 15E Pr-to-15Z Pg conversion with a significant solvent-isotope effect, suggesting the sequential occurrence of 15EP-to-15Z photoisomerization, deprotonation, and second thiol adduct formation. Our findings uncover the details of protochromic absorption changes underlying the two-Cys photocycle of violet-blueabsorbing CBCR subfamilies. Phytochromes are photosensors that bind a linear tetrapyrrole (bilin) chromophore and are found in higher plant, algae, bacteria, and fungi (1-3). They undergo reversible photoconversion typically between the red-absorbing state (Pr) and the far-red light-absorbing state (Pfr), 2 which is triggered by the 15Z/15E photoisomerization of the bilin (Fig. S1) (4). In the dark, phytochromes convert to thermally stable Pr or Pfr via a process called dark reversion. The bilin is bound to the pocket of a GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domain via a conserved Cys residue that is found in the GAF domain for phycocyanobilin (PCB) and phytochromobilin (Fig. 1A, canonical Cys) or in the N terminus for biliverdin (5). The GAF domain interacts with the phytochrome domain via tongue structure and, in many cases, with the Per/Arnt/Sim (PAS) domain with the knot structure (6-8). These domains are required to complete the photocycle of phytochromes. Cyanobacteriochromes (CBCRs) are unique phytochromerelated photosensors that are confined to but widely distributed among cyanobacteria (2, 9-14)....

Photocycle of Cyanobacteriochrome TePixJ

“… 24 , 25 , 30 , 31 It has also been hypothesized that deprotonation of the bilin is required to promote the interaction with the neutral thiol group during the formation of the second Cys linkage. 32 The transition from EADS4 to EADS5 displays a moderate kinetic isotope effect (KIE, lifetimes of 18.7 ns in D 2 O and 11.4 ns in H 2 O), making this a plausible proton transfer step. However, such a step should have a distinct infrared spectral signature, 25 and blue-shifted absorbance maxima, 14 , 33 neither of which are observed here, so the observed KIE may be due to only changed dynamics due to exchangeable protons making the protein “heavier” and transitions slower.…”

Spectral and photochemical diversity of tandem cysteine cyanobacterial phytochromes

“…To test whether the varying ratio of YO-and Rabsorbing photoproducts is due to differences in ionization states of the chromophore and/or nearby amino acid residues, we undertook experiments to test the effect of pH on the ToTCCP photoproduct spectrum -an approach was used previously to examine the pHdependence of both dark and photoproduct states of the G/R CBCR RcaE (29) and the B/O CBCR Oscil6304_2705 (26). As expected for a neutral doubly-linked chromophore (Fig.…”

“…Multiple dual-Cys CBCR lineages have been characterized to date (15,16,(19)(20)(21)(22)(23)(24)(25)(26). However, the tandem-Cys trichromatic phytochrome NpTP1 encoded by gene locus NpunF1183 from Nostoc punctiforme ATCC 29133 is the only dual-Cys cyanobacterial phytochrome to have been investigated at the biochemical level (15).…”

Structural basis of the protochromic green/red photocycle of the chromatic acclimation sensor RcaE

“…In this mechanism, a change in the protonation state of the bilin pyrrole system is responsible for the change in absorption. The deprotonation-induced formation of green-absorbing state has also been observed as an intermediate in the two-Cys photocycle (19). The red/green subfamily harbors a fully protonated chromophore in both photostates, but a twisted conformation of the A-and Drings is responsible for the blue-shifted photoproduct (8,(20)(21)(22)(23); this is known as the "trappedtwist photocycle" (24).…”