Electronic transitions and heterogeneity of the bacteriophytochrome Pr absorption band: An angle balanced polarization resolved femtosecond VIS pump–IR probe study

Abstract

Photoisomerization of biliverdin (BV) chromophore triggers the photoresponse in native Agp1 bacteriophytochrome. We discuss heterogeneity in phytochrome Pr form to account for the shape of the absorption profile. We investigated different regions of the absorption profile by angle balanced polarization resolved femtosecond VIS pump-IR probe spectroscopy. We studied the Pr form of Agp1 with its natural chromophore and with a sterically locked 18Et-BV (locked Agp1). We followed the dynamics and orientations of the carbonyl stretching vibrations of ring D and ring A in their ground and electronically excited states. Photoisomerization of ring D is reflected by strong signals of the ring D carbonyl vibration. In contrast, orientational data on ring A show no rotation of ring A upon photoexcitation. Orientational data allow excluding a ZZZasa geometry and corroborates a nontwisted ZZZssa geometry of the chromophore. We found no proof for heterogeneity but identified a new, to our knowledge, electronic transition in the absorption profile at 644 nm (S0→S2). Excitation of the S0→S2 transition will introduce a more complex photodynamics compared with S0→S1 transition. Our approach provides fundamental information on disentanglement of absorption profiles, identification of chromophore structures, and determination of molecular groups involved in the photoisomerization process of photoreceptors.

Spectroscopic Investigation on the Primary Photoreaction of Bathy Phytochrome Agp2‐Pr of Agrobacterium fabrum: Isomerization in a pH‐dependent H‐bond Network

“…These results thus indicate rather well defined molecular states and only little ground‐state heterogeneity concerning the keto/enol equilibrium at the chosen pH values. On the other hand, at this point we cannot exclude further structural inhomogeneity as, for example, described for the Pr primary reaction in Cph1 and Agp1 and discussed for Pa BphP …”

“…Besides observed KIEs af urtheri mportant aspect for the construction of reaction schemes concerns structuralo rd y-namic inhomogeneities as for example, described for the Pr primaryr eaction in Cph1 [31,53,63] and Agp1 [64] and discussed for PaBphP. [48] In particularf or Cph1 [31] and RpBphP2 [25] the electronic deactivation via parallel reaction channels originating Figure 5.…”

Influence of Heterogeneity on the Ultrafast Photoisomerization Dynamics of Pfr in Cph1 Phytochrome

“…Because electronic state dynamics of phytochromes are spectrally complex, we analyzed the system by combining electronic state dynamics measured by femtosecond (fs) visible (Vis) pump–supercontinuum Vis probe spectroscopy with angle‐balanced polarization‐resolved femtosecond Vis pump–infrared (IR) probe spectroscopy . This allows us to separate electronic dynamics from photoproduct generation and hot ground‐state relaxation . Ultrafast IR spectroscopy is an ideal tool to identify the appearance of photoproduct bands upon photoexcitation .…”

Non‐Bonded Interactions Drive the Sub‐Picosecond Bilin Photoisomerization in the Pfr State of Phytochrome Cph1

“…Next we applied angle-balanced femtosecondp olarizationresolved VIS pump IR probe spectroscopy. [13] After excitingt he unlabeled chromophore within the 13 C/ 15 Nl abeledp rotein sample at 710 nm, we probedt he polarization resolved transient signals in as pectral range from 1660 to 1750 cm À1 . [6a] In Figure 4, isotropic vibrational difference spectra for different delay times are presented.…”

“…[14] We assign the spectrally narrow structured features visible at 1675, 1685, 1694, and 1733 cm À1 in the first hundred femtoseconds to water vapor absorption. [13] The bleaching band of the C 1 =Os tretching vibration n(C 1 =O) is expected to show up around 1725 cm À1 ,a long with the C 19 =Os tretching vibration n(C 19 =O) LF of the first photoproduct Lumi-F. [13, 6a] This is reflected in the absorbance difference spectra,w here the instantaneous negative bleaching signal of the n(C 1 =O) vibration dominates at early delay times, while after 1ps, the signalb ecomes positive due to decreasing bleaching signal and increasingL umi-F photoproduct absorption. For longerd elay times, the n(C 1 =O) bleaching band vanishes completelyw ith the decay of the electronic excited state.…”