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

By Marc G. Müller, Ingo Lindner, Iris Martin, Wolfgang Gärtner and Alfred R. Holzwarth


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 (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 ∼690 nm, is mainly described by 28 ps or 40 ps components in native and PCB phytochrome, respectively, whereas additional ∼15 and 50 ps 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-65 kDa-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 ∼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 ∼400 ps 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-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 ∼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

Topics: Photobiophysics
Publisher: The Biophysical Society
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Provided by: PubMed Central
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