Comparison of the Forward and Reverse Photocycle Dynamics of Two Highly Similar Canonical Red/Green Cyanobacteriochromes Reveals Unexpected Differences

Abstract

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors that exhibit photochromism between two states: a thermally stable dark-adapted state and a metastable light-adapted state with bound linear tetrapyrrole (bilin) chromophores possessing 15Z and 15E configurations, respectively. The photodynamics of canonical red/green CBCRs have been extensively studied; however, the time scales of their excited-state lifetimes and subsequent ground-state evolution rates widely differ and, at present, remain difficult to predict. Here, we compare the photodynamics of two closely related red/green CBCRs that have substantial sequence identity (∼68%) and similar chromophore environments: AnPixJg2 from Anabaena sp. PCC 7120 and NpR6012g4 from Nostoc punctiforme. Using broadband transient absorption spectroscopy on the primary (125 fs to 7 ns) and secondary (7 ns to 10 ms) time scales together with global analysis modeling, our studies revealed that AnPixJg2 and NpR6012g4 have comparable quantum yields for initiating the forward ( 15Z P r → 15E P g ) and reverse ( 15E P g → 15Z P r ) reactions, which proceed through monotonic and nonmonotonic mechanisms, respectively. In addition to small discrepancies in the kinetics, the secondary reverse dynamics resolved unique features for each domain: intermediate shunts in NpR6012g4 and a Meta-G f intermediate redshifted from the 15Z P r photoproduct in AnPixJg2. Overall, this study supports the conclusion that sequence similarity is a useful criterion for predicting pathways of the light-induced evolution and quantum yield of generating primary intermediate Φ p within subfamilies of CBCRs, but more studies are still needed to develop a comprehensive molecular level understanding of these processes.

Needles in a haystack: H-bonding in an optogenetic protein observed with isotope labeling and 2D-IR spectroscopy

“…On average, these interactions have the same effect on the carbonyl as the single H-bond to the Tyr residue in Pr, which accounts for the same central peak position. In the difference-FTIR spectra, this broadening in a heterogeneous environment is reflected by a sharp negative contribution at 1674 cm −1 on top of a broad positive background, which was also observed at similar frequencies in the difference FTIR spectra of AnpixJg2, 35,36 NpR6012g4 37 and Slr1393g3. 25 …”

The impact of chromophore choice on the assembly kinetics and primary photochemistry of a red/green cyanobacteriochrome

“…55%). 27,31,39 AmI-g2 ranges on the lower end of the spectrum for the Φ L in both directions, with values < 10% and amplitudes comparable e.g. to Lumi-R formation in Slr1393-g3.…”

An Engineered Biliverdin-Compatible Cyanobacteriochrome Enables a Unique Ultrafast Reversible Photoswitching Pathway

“…In addition, an expanded spectroscopic toolset from ultrafast regime (typically fs-to-ns time scales as reported here) to the more macroscopic time scales (ns-to-ms or longer) could better identify long-lived or later intermediates (e.g., Lumi-G and Lumi-R in Apcb and Bpcb, and Lumi-O and Lumi-Fr in Bbv), which may dictate the forward/reverse reaction speed and yield [ 5 , 29 , 60 , 61 ]. These Lumi states are likely similar to the photoproduct species in our current experiments on the ns time scale ( Figure 5 ), however, they require further protein relaxation and chromophore pocket rearrangement on the electronic ground state to eventually reach the final product state.…”

“…These Lumi states are likely similar to the photoproduct species in our current experiments on the ns time scale ( Figure 5 ), however, they require further protein relaxation and chromophore pocket rearrangement on the electronic ground state to eventually reach the final product state. For example, a TA setup with pulsed (100 ns) LED-based broadband probe has expanded the time window from ns to ms and identified two intermediates between Lumi-G 2 and P r after 532 nm excitation of P g of NpR6012g4 [ 62 ], and recent applications in conjunction with fs-TA were able to elucidate a series of meta states that emerge after the Lumi state formation in AnPixJg2 as well as NpR6012g4 with a strong sequence homology (both are canonical red/green CBCRs) [ 61 ]. In aggregate, by combining multiple techniques such as TA, FSRS, QM/MM, X-ray crystallography, and NMR, the resultant comprehensive information can delineate the complex photoconversion of CBCRs and other photosensing proteins as they convert from one state to another on various time scales.…”