Phytochrome as Molecular Machine: Revealing Chromophore Action during the Pfr → Pr Photoconversion by Magic-Angle Spinning NMR Spectroscopy

Abstract

The cyanobacterial phytochrome Cph1 can be photoconverted between two thermally stable states, Pr and Pfr. The photochemically induced Pfr --> Pr back-reaction has been followed at low temperature by magic-angle spinning (MAS) NMR spectroscopy, allowing two intermediates, Lumi-F and Meta-F, to be trapped. Employing uniformly (13)C- and (15)N-labeled open-chain tetrapyrrole chromophores, all four states-Pfr, Lumi-F, Meta-F, and Pr-have been structurally characterized. In the first step, the double bond photoisomerization forming Lumi-F occurs. The second step, the transformation to Meta-F, is driven by the release of the mechanical tension. This process leads to the break of the hydrogen bond of the ring D nitrogen to Asp-207 and triggers signaling. The third step is protonically driven allowing the hydrogen-bonding interaction of the ring D nitrogen to be restored. Compared to the forward reaction, the order of events is changed, probably caused by the different properties of the hydrogen bonding partners of N24, leading to the directionality of the photocycle.

Structure-guided Engineering Enhances a Phytochrome-based Infrared Fluorescent Protein

“…The same interaction has been suggested for the Pfr structure of the cyanobacterial Cph1 (23,34). In our DrCBD-D207H H:Aring structure, the His-207 rotamer can no longer stabilize this Pfr chromophore conformation (Figs.…”

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

“…The Pr → Pfr photoflip breaks the interaction between ring D and His-290 and leads to the formation of hydrogen-bonding interactions of the D-ring carbonyl group to Tyr-263 and of N24 to Asp-207 (Figs. 1E and 4C), apparently stabilizing the Pfr chromophore in a more tensed conformation (33), as also implied by the PaBphP 3C2W Pfr structure (22). The newly formed Pfr interactions (Tyr-263⋯C19 and Asp-207⋯N24) require disruption of the Asp-207/Arg-472 salt bridge (Fig.…”

The D-ring, Not the A-ring, Rotates in Synechococcus OS-B′ Phytochrome

“…By way of comparison, the D-ring photoflip in the canonical Cph1 and plant phytochromes is associated with striking ␦ C changes of ϳ6 ppm for C14 and most D-ring pyrrolic carbons (see Table 1). Similarly, however, there is also little support for C4ϭC5 isomerization and the proposed A-ring photoflip (24); the C4 and C6 atoms associated with the A-B methine bridge show a ␦ C change less than 1.3 ppm, far smaller than that from C14 and C16 of the C-D bridge associated with the D-ring photoflip in canonical phytochromes (13,17,18). Furthermore, ␦ C changes seen at ring A (C3 (Ϫ2.3/Ϫ0.9 ppm), its side chain C3 1 and C3 2 (ϩ1.0 and Ϫ1.3 ppm, respectively) linkage to Cys-138, and pyrrolic car-bons C1 (ϩ1.3 ppm) and C2 (ϩ1.2 ppm)) are also too small for an A-ring photoflip.…”