Proton-transfer and hydrogen-bond interactions determine fluorescence quantum yield and photochemical efficiency of bacteriophytochrome

Abstract

Phytochromes are red-light photoreceptor proteins that regulate a variety of responses and cellular processes in plants, bacteria, and fungi. The phytochrome light activation mechanism involves isomerization around the C15═C16 double bond of an open-chain tetrapyrrole chromophore, resulting in a flip of its D-ring. In an important new development, bacteriophytochrome (Bph) has been engineered for use as a fluorescent marker in mammalian tissues. Here we report that an unusual Bph, RpBphP3 from Rhodopseudomonas palustris, denoted P3, is fluorescent. This Bph modulates synthesis of light-harvesting complex in combination with a second Bph exhibiting classical photochemistry, RpBphP2, denoted P2. We identify the factors that determine the fluorescence and isomerization quantum yields through the application of ultrafast spectroscopy to wild-type and mutants of P2 and P3. The excited-state lifetime of the biliverdin chromophore in P3 was significantly longer at 330-500 ps than in P2 and other classical phytochromes and accompanied by a significantly reduced isomerization quantum yield. H/D exchange reduces the rate of decay from the excited state of biliverdin by a factor of 1.4 and increases the isomerization quantum yield. Comparison of the properties of the P2 and P3 variants shows that the quantum yields of fluorescence and isomerization are determined by excited-state deprotonation of biliverdin at the pyrrole rings, in competition with hydrogen-bond rupture between the D-ring and the apoprotein. This work provides a basis for structure-based conversion of Bph into an efficient near-IR fluorescent marker.hydrogen bonds | near-IR fluorescent protein | reaction mechanism | kinetic isotope effect | biophotonics

Two ground state isoforms and a chromophore D -ring photoflip triggering extensive intramolecular changes in a canonical phytochrome

“…Also, ultrafast absorption spectroscopy on wild-type and mutants of Rp. palustris RpBphP2 showed two kinetic components (39). Discrepancies between Pr fluorescence excitation and absorbance spectra of Cph1Δ2 have also been noted and associated with Pr heterogeneity (40).…”

Fluorescence of Phytochrome Adducts with Synthetic Locked Chromophores

“…In wild type Agp1, Asp-197 is part of a hydrogenbonding network that involves rings B and C of the chromophore (7,11,31). Disturbing this network by an Asp-197 replacement could also impact the hydrogen-bonding network around ring D and, in this way, inhibit the energy dissipation pathway proposed above (56).…”

“…It is unclear whether inside the protein this flexibility is high enough to provide an energy dissipation pathway for Ͼ60% of absorbed photons. (ii) Excited state proton transfer: In a recent publication on bacteriophytochrome BphP3 from R. palustris, it has been put forward that the excited state of phytochrome might be deactivated by a rapid (350 ps) proton transfer that finally ends up in a back-conversion to Pr (56). Such a reaction would compete with fluorescence that has a lifetime in the ns range (22).…”

Tracking the secondary photodynamics of the green/red cyanobacteriochrome RcaE from Fremyella diplosiphon

“…In the primary (100 fs-10 ns) photodynamics of most phytochromes [17][18][19][20][21][22] and CBCRs [22][23][24][25][26][27][28] characterized to date, a single primary intermediate is typically resolved after the photoexcitation of the respective dark-adapted states. However, forward reaction photodynamics of RcaE are unique, with three primary intermediates observed after excitation of…”