Abstract
Phytochromes are biological photoreceptors that can be reversibly photoconverted between a dark and photoactivated state. The underlying reaction sequences are initiated by the photoisomerization of the tetrapyrrole cofactor, which in plant and cyanobacterial phytochromes are a phytochromobilin (PΦB) and a phycocyanobilin (PCB), respectively. The transition between the two states represents an on/off-switch of the output module activating or deactivating downstream physiological processes. In addition, the photoactivated state, i.e., Pfr in canonical phytochromes, can be thermally reverted to the dark state (Pr). The present study aimed to improve our understanding of the specific reactivity of various PΦB- and PCB-binding phytochromes in the Pfr state by analysing the cofactor structure by vibrational spectroscopic techniques. Resonance Raman (RR) spectroscopy revealed two Pfr conformers (Pfr-I and Pfr-II) forming a temperature-dependent conformational equilibrium. The two sub-states—found in all phytochromes studied, albeit with different relative contributions—differ in structural details of the C-D and A-B methine bridges. In the Pfr-I sub-state the torsion between the rings C and D is larger by ca. 10° compared to Pfr-II. This structural difference is presumably related to different hydrogen bonding interactions of ring D as revealed by time-resolved IR spectroscopic studies of the cyanobacterial phytochrome Cph1. The transitions between the two sub-states are evidently too fast (i.e., nanosecond time scale) to be resolved by NMR spectroscopy which could not detect a structural heterogeneity of the chromophore in Pfr. The implications of the present findings for the dark reversion of the Pfr state are discussed.
Highlights
Phytochromes are ubiquitous photoreceptor in plants that utilize light as a source of information for controlling photomorphogenic processes (Quail, 1998; Schäfer and Nagy, 2006)
We have studied a variant of the bacterial phytochrome Agp1, Agp1-V249C, in which the natural BV attachment site was by one at position 249 to allow for binding of PCB (Borucki et al, 2009)
The present study has demonstrated that the Pfr states of a number of PCB- and P B-binding phytochromes display a structural heterogeneity of the chromophore involving two main sub-states, differing at the A-B and C-D methine bridges
Summary
Phytochromes are ubiquitous photoreceptor in plants that utilize light as a source of information for controlling photomorphogenic processes (Quail, 1998; Schäfer and Nagy, 2006). Upon light excitation phytochromes are interconverted between the red-absorbing (Pr) and far-red absorbing state (Pfr), corresponding to a switch between physiologically inactive and active states, respectively. The primary photochemical step of the Pr → Pfr conversion is a double-bond isomerization (Z/E) of the C-D methine bridge (Rockwell et al, 2006; Rockwell and Lagarias, 2010). The resting state of the small “bathy”-type group is Pfr rather than Pr. Here dark reversion from Pr to Pfr takes place, corresponding to a thermal E → Z double bond isomerization that is initiated by a keto/enol tautomerization (Velazquez Escobar et al, 2015). It might well be that an analogous mechanism holds for Pfr → Pr dark reversion in canonical phytochromes as suggested earlier (Lagarias and Rapoport, 1980)
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