Solid-state NMR spectroscopy to probe photoactivation in canonical phytochromes

The photoreceptor phytochrome switches photochromically between two thermally stable states called Pr and Pfr. Here, we summarize recent solid-state magic-angle spinning (MAS) NMR work on this conversion process and interpret the functional mechanism in terms of a nano-machine. The process is initiated by a double-bond photoisomerization of the open-chain tetrapyrrole chromophore at the methine bridge connecting pyrrole rings C and D. The Pr-state chromophore and its surrounding pocket in canonical cyanobacterial and plant phytochromes has significantly less order, tends to form isoforms and is soft. Conversely, Pfr shows significantly harder chromophore-protein interactions, a well-defined protonic and charge distribution with a clear classical counterion for the positively charged tetrapyrrole system. The soft-to-hard/disorder-to-order transition involves the chromophore and its protein surroundings within a sphere of at least 5.5 Å. The relevance of this collective event for signaling is discussed. Measurement of the intermediates during the Pfr → Pr back-reaction provides insight into the well-adjusted mechanics of a two-step transformation. As both Pr → Pfr and Pfr → Pr reaction pathways are different in ground and excited states, a photochemically controlled hyper-landscape is proposed allowing for ratchet-type reaction dynamics regulating signaling activity.