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

AbstractThe photoprocesses of native (phyA of oat), and of C-terminally truncated recombinant phytochromes, assembled instead of the native phytochromobilin with phycocyanobilin (PCB-65kDa-phy) and iso-phycocyanobilin (iso-PCB-65kDa-phy) chromophores, have been studied by femtosecond transient absorption spectroscopy in both their red absorbing phytochrome (Pr) and far-red absorbing phytochrome (Pfr) forms. Native Pr phytochrome shows an excitation wavelength dependence of the kinetics with three main picosecond components. The formation kinetics of the first ground-state intermediate I700, absorbing at ∼690nm, is mainly described by 28ps or 40ps components in native and PCB phytochrome, respectively, whereas additional ∼15 and 50ps components describe conformational dynamics and equilibria among different local minima on the excited-state hypersurface. No significant amount of I700 formation can be observed on our timescale for iso-PCB phytochrome. We suggest that iso-PCB-65kDa-phy either interacts with the protein differently leading to a more twisted and/or less protonated configuration, or undergoes Pr to Pfr isomerization primarily via a different configurational pathway, largely circumventing I700 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 Pfr forms is again quite similar for the native and the PCB phytochromes. After an ultrafast excited-state relaxation within ∼150fs, the chromophores return to the first ground-state intermediate in 400–800fs followed by two additional ground-state intermediates which are formed with 2–3ps and ∼400ps lifetimes. We call the first ground-state intermediate in native phytochrome Ifr·750, due to its pronounced absorption at that wavelength. The other intermediates are termed Ifr·675 and pseudo-Pr. The absorption spectrum of the latter already closely resembles the absorption of the Pr chromophore. PCB-65kDa-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-65kDa-phy shows similar features to the PCB-65kDa-phy, with some additional blue-shift of the transient spectra of ∼10nm. The sub-200fs component is, however, absent, and the picosecond lifetimes are somewhat longer than in 124kDa phytochrome or in PCB-65kDa-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