Femtosecond Photodynamics of the Red/Green Cyanobacteriochrome NpR6012g4 from Nostoc punctiforme. 2. Reverse Dynamics

Abstract

Phytochromes are red/far-red photosensory proteins that utilize photoisomerization of a linear tetrapyrrole (bilin) chromophore to photoconvert reversibly between red- and far-red-absorbing forms (P(r) and P(fr), respectively). Cyanobacteriochromes (CBCRs) are related photosensory proteins with more diverse spectral sensitivity. The mechanisms that underlie this spectral diversity have not yet been fully elucidated. One of the main CBCR subfamilies photoconverts between a red-absorbing 15Z ground state, like the familiar P(r) state of phytochromes, and a green-absorbing photoproduct ((15E)P(g)). We have previously used the red/green CBCR NpR6012g4 from the cyanobacterium Nostoc punctiforme to examine ultrafast photodynamics of the forward photoreaction. Here, we examine the reverse reaction. Using excitation-interleaved transient absorption spectroscopy with broadband detection and multicomponent global analysis, we observed multiphasic excited-state dynamics. Interleaved excitation allowed us to identify wavelength-dependent shifts in the ground-state bleach that equilibrated on a 200 ps time scale, indicating ground-state heterogeneity. Compared to the previously studied forward reaction, the reverse reaction has much faster excited-state decay time constants and significantly higher photoproduct yield. This work thus demonstrates striking differences between the forward and reverse reactions of NpR6012g4 and provides clear evidence of ground-state heterogeneity in the phytochrome superfamily.

Dynamic Inhomogeneity in the Photodynamics of Cyanobacterial Phytochrome Cph1

“…Thus, the DEWI-PP approach ensures that the primary experimental variable is the excitation energy, which was successfully utilized previously to study excitation-dependent dynamics in carotenoids and phytochrome-related CBCRs. 10 …”

Characterization of Red/Green Cyanobacteriochrome NpR6012g4 by Solution Nuclear Magnetic Resonance Spectroscopy: A Protonated Bilin Ring System in Both Photostates

“…It was thus possible that we were detecting two configurations for a single propionate side chain rather than the expected 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 25 pair of side chains, in keeping with the known photochemical heterogeneity of NpR6012g4. 52,[54][55][56][57] We therefore acquired three-dimensional HCCH-TOCSY spectra (Figs. 9 & S4).…”

“…51 However, AnPixJg2 and other red/green CBCRs are known to be heterogeneous. 43,[52][53][54][55][56][57][58] It is thus possible that this heterogeneity could confound interpretation of the vibrational spectra. A detailed analysis of the Resonance Raman studies in combination with molecular dynamics simulations led to the proposal that the photoproduct of AnPixJg2 is extensively solvated due to movement of a conserved 'lid'…”

“…Red/green CBCRs are typically characterized at pH 7 -pH 8.5. 38,39,43,[52][53][54][55][56][57][60][61][62] One would thus expect formation of a deprotonated, neutral bilin π system in the photoproduct under the solvation model (Fig. S2B).…”

Structures of cyanobacteriochromes from phototaxis regulators AnPixJ and TePixJ reveal general and specific photoconversion mechanism

“…The red/green-type CBCRs represented by AnPixJ (PixJ of Anabaena sp. PCC 7120) show photoconversion between redabsorbing 15Z and green-absorbing 15E (Pg) forms (27)(28)(29)(30)(31)(32). In this type, it is suggested that the Pr form is almost equivalent to that of the phytochromes and starts a primary photoreaction with Z-to-E isomerization in a mechanism similar to that in the phytochromes, but is finally photoconverted to the unique Pg form (27,29,31).…”