Femtosecond Kinetics of Photoconversion of the Higher Plant Photoreceptor Phytochrome Carrying Native and Modified Chromophores

Abstract

The photoprocesses of native (phyA of oat), and of C-terminally truncated recombinant phytochromes, assembled instead of the native phytochromobilin with phycocyanobilin (PCB-65 kDa-phy) and iso-phycocyanobilin (iso-PCB-65 kDa-phy) chromophores, have been studied by femtosecond transient absorption spectroscopy in both their red absorbing phytochrome (P(r)) and far-red absorbing phytochrome (P(fr)) forms. Native P(r) phytochrome shows an excitation wavelength dependence of the kinetics with three main picosecond components. The formation kinetics of the first ground-state intermediate I(700), absorbing at approximately 690 nm, is mainly described by 28 ps or 40 ps components in native and PCB phytochrome, respectively, whereas additional approximately 15 and 50 ps components describe conformational dynamics and equilibria among different local minima on the excited-state hypersurface. No significant amount of I(700) formation can be observed on our timescale for iso-PCB phytochrome. We suggest that iso-PCB-65 kDa-phy either interacts with the protein differently leading to a more twisted and/or less protonated configuration, or undergoes P(r) to P(fr) isomerization primarily via a different configurational pathway, largely circumventing I(700) as an intermediate. The isomerization process is accompanied by strong coherent oscillations due to wavepacket motion on the excited-state surface for both phytochrome forms. The femto- to (sub-)nanosecond kinetics of the P(fr) forms is again quite similar for the native and the PCB phytochromes. After an ultrafast excited-state relaxation within approximately 150 fs, the chromophores return to the first ground-state intermediate in 400-800 fs followed by two additional ground-state intermediates which are formed with 2-3 ps and approximately 400 ps lifetimes. We call the first ground-state intermediate in native phytochrome I(fr 750), due to its pronounced absorption at that wavelength. The other intermediates are termed I(fr 675) and pseudo-P(r). The absorption spectrum of the latter already closely resembles the absorption of the P(r) chromophore. PCB-65 kDa-phy shows a very similar kinetics, although many of the detailed spectral features in the transients seen in native phy are blurred, presumably due to wider inhomogeneous distribution of the chromophore conformation. Iso-PCB-65 kDa-phy shows similar features to the PCB-65 kDa-phy, with some additional blue-shift of the transient spectra of approximately 10 nm. The sub-200 fs component is, however, absent, and the picosecond lifetimes are somewhat longer than in 124 kDa phytochrome or in PCB-65 kDa-phy. We interpret the data within the framework of two- and three-dimensional potential energy surface diagrams for the photoisomerization processes and the ground-state intermediates involved in the two photoconversions.

Ultrafast excited-state isomerization in phytochrome revealed by femtosecond stimulated Raman spectroscopy

“…The 26-ps decay of the stimulated emission (840-960 nm) is consistent with the 28-ps excited-state lifetime attributed to the P r * excited state from fluorescence quantum yield analysis (14). The 30-ps formation of Lumi-R is also in agreement with time-resolved transient absorption experiments on native and substituted chromophores in PhyA and Cph1 phytochromes (7,11). Therefore, the stimulated emission decay dynamics, together with the changes in intensity of the Raman bands, directly indicate that the 3-ps Z-to-E isomerization takes place on the long-lived excited state and that the ground-state Lumi-R photoproduct forms in 30 ps.…”

“…6 presents a schematic potential energy surface summarizing the structural changes we have observed in the P r -to-P fr photoreaction of Cph1. After excitation, the system moves away from the FC region within 150 fs indicated by the rise of RINE signals (8,11). The decay of the RINE signals marks the transition to the excitedstate I* with a highly distorted chromophore structure in the C 14 OC 15 AC 16 region.…”

“…The P r excited state exhibits multiexponential fluorescence decay dynamics with at least two lifetimes (10 ps and 45 ps) (5), thereby implicating the presence of at least two excited states. The two-state model for P r * decay has also received support from ultrafast transient absorption measurements on the plant phytochrome phyA, the cyanobacterial phytochrome Cph1, and the bacteriophytochrome Agp1, all of which exhibit two excited-state lifetimes characterized by a short (5-16 ps) and a long (25-40 ps) time constant (8,10,11). Despite the kinetic insight of these transient electronic spectroscopic studies, these techniques do not establish when the double bond isomerization occurs, nor do they resolve the molecular basis for the low P r -to-P fr photochemical quantum yields (7-16%) (12,13).…”

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…”

Phototransformation of the Red Light Sensor Cyanobacterial Phytochrome 2 from Synechocystis Species Depends on Its Tongue Motifs

“…The further steps are light-independent and proceed thermally driven in longer time scales. Accordingly, phytochrome photoconversion starts within picoseconds and can last up to seconds (2,3). The light-triggered reaction from P fr 3 P r undergoes different intermediates (4,5).…”