Forward Photodynamics of the Noncanonical Red/Green NpR3784 Cyanobacteriochrome from Nostoc punctiforme

Abstract

Cyanobacteriochromes (CBCRs) make up a diverse family of cyanobacterial photoreceptors distantly related to the phytochrome photoreceptors of land plants. At least two lineages of CBCRs have reacquired red-absorbing dark states similar to the phytochrome P r resting state but are coupled to greenabsorbing light-adapted states rather than the canonical far-red-absorbing lightadapted state. One such lineage includes the canonical red/green (R/G) CBCRs that includes AnPixJg2 (UniProtKB Q8YXY7) and NpR6012g4 (UniProtKB B2IU14) that have been extensively characterized. Here we examine the forward P r photodynamics of NpR3784 (UniProtKB B2J457), a representative member of the second R/G CBCR subfamily. Using broadband transient absorption pump−probe spectroscopy, we characterize both primary (100 fs to 10 ns) and secondary (10 ns to 1 ms) forward (P r → P g ) photodynamics and compare the results to temperature-jump cryokinetics measurements. Our studies show that primary isomerization dynamics occur on an ∼10 ps timescale, yet remarkably, the redshifted primary Lumi-R f photoproduct found in all photoactive canonical R/G CBCRs examined to date is extremely short-lived in NpR3784. These results demonstrate that differences in reaction pathways reflect the evolutionary history of R/G CBCRs despite the convergent evolution of their photocycle end products.

Reverse Photodynamics of the Noncanonical Red/Green NpR3784 Cyanobacteriochrome from Nostoc punctiforme

“…From the present and previous studies, the observed multiphasic decay profiles of the primary excited-state species in both directions are consistent with ground-state heterogeneity. In the forward direction, the three excited-state populations evolve on different time scales via similar spectral intermediates, that is, Lumi-R f , Meta-R o , Meta-R y , and Meta-R g , although the primary photoproducts were observable only for the fastest population at room temperature and by low-temperature trapping . By contrast, the two excited-state populations produced in the reverse direction yield spectrally distinct Lumi photoproducts, one of which evolves to the 15 Z P r dark state, whereas the other slowly repopulates the former.…”

“…In contrast with the forward reaction dynamics previously described, the regular approach for generating the light-induced state in aqueous buffer by illumination does not fully convert the protein population into the light 15 E P g state in cryosolvent. The 650 nm photoexcitation of the cryosample in the 15 Z P r at 230 K for 1.5 h does not generate the pure 15 E P g state observed in the room-temperature excitation of NpR3784 in an aqueous buffer (Figure ).…”

“…After cooling to 160 K, the sample was illuminated for 2 h to initiate the reverse ( 15 E P g → 15 Z P r ) photocycle with laser diodes of three excitation wavelengths (510, 530, or 585 nm). Cryokinetics data were analyzed with the same global analysis routines (previously discussed) used for room-temperature kinetics analyses.…”

“…PCC6803. ,− All of these share similar excited-state photochemistries and subsequent ground-state protein dynamics with phytochromes in the forward direction (Scheme ). ,,− In the previous study, the forward 15 Z P r → 15 E P g photocycle of the noncanonical R/G CBCR NpF3784 was examined . That study revealed that the 15 Z P r ground state is composed of three ground-state populations that generate primary photoproducts.…”

Nanosecond protein dynamics in a red/green cyanobacteriochrome revealed by transient IR spectroscopy

“…The region in between (550-620 nm) contains overlapping contributions from ESA and GSB. Similar excited-state spectra were also obtained for several related red/green CBCRs from Nostoc punctiforme, albeit with large variations in lifetimes [41][42][43][44]. In the lifetime analysis of Slr-g3, a local maximum of D is observed at 600 ps, and global fitting resulted in an optimized time constant of 540 ps (τ 1 , see table I).…”