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

Background: Phytochrome photoreceptors are activated by light-induced isomerization of the chromophore cofactor. Results: Photoactivation of Synechococcus OS-B phytochrome breaks an unusual chromophore D-ring hydrogen bond, whereas only subtle changes occur at the A-ring linkage to the protein. Conclusion: Activation arises from a photoflip of a strongly tilted D-ring. Significance: The hypothesis that the A-ring rotates upon photon absorption is wrong. Phytochrome photoreceptors in plants and microorganisms switch photochromically between two states, controlling numerous important biological processes. Although this phototransformation is generally considered to involve rotation of ring D of the tetrapyrrole chromophore, Ulijasz et al. (Ulijasz, A. T., Cornilescu, G., Cornilescu, C. C., Zhang, J., Rivera, M., Markley, J. L., and Vierstra, R. D. (2010) Nature 463, 250-254) proposed that the A-ring rotates instead. Here, we apply magic angle spinning NMR to the two parent states following studies of the 23-kDa GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domain fragment of phytochrome from Synechococcus OS-B. Major changes occur at the A-ring covalent linkage to the protein as well as at the protein residue contact of ring D. Conserved contacts associated with the A-ring nitrogen rule out an A-ring photoflip, whereas loss of contact of the D-ring nitrogen to the protein implies movement of ring D. Although none of the methine bridges showed a chemical shift change comparable with those characteristic of the D-ring photoflip in canonical phytochromes, denaturation experiments showed conclusively that the same occurs in Synechococcus OS-B phytochrome upon photoconversion. The results are consistent with the D-ring being strongly tilted in both states and the C15=C16 double bond undergoing a Z/E isomerization upon light absorption. More subtle changes are associated with the A-ring linkage to the protein. Our findings thus disprove A-ring rotation and are discussed in relation to the position of the D-ring, photoisomerization, and photochromicity in the phytochrome family.