Chromophore structure in the photocycle of the cyanobacterial phytochrome Cph1

The chromophore conformations of the red and far red light induced product states Pfr'' and Pr'' of the N-terminal photoreceptor domain Cph1-N515 from Synechocystis 6803 have been investigated by NMR spectroscopy, using specific C-13 isotope substitutions in the chromophore. C-13-NMR spectroscopy in the Pfr and Pr states indicated reversible chemical shift differences predominantly of the C-4 carbon in ring A of the phycocyanobilin chromophore, in contrast to differences of C-15 and C-5, which were much less pronounced. Ab initio calculations of the isotropic shielding and optical transition energies identify a region for C-4-C-5-C-6-N-2 dihedral angle changes where deshielding of C-4 is correlated with red-shifted absorption. These could occur during thermal reactions on microsecond and millisecond timescales after excitation of Pr which are associated with red-shifted absorption. A reaction pathway involving a hula-twist at C-5 could satisfy the observed NMR and visible absorption changes. Alternatively, C-15 Z-E photoisomerization, although expected to lead to a small change of the chemical shift of C-15, in addition to changes of the C-4-C-5-C-6-N-2 dihedral angle could be consistent with visible absorption changes and the chemical shift difference at C-4. NMR spectroscopy of a C-13-labeled chromopeptide provided indication for broadening due to conformational exchange reactions in the intact photoreceptor domain, which is more pronounced for the C- and D-rings of the chromophore. This broadening was also evident in the F2 hydrogen dimension from heteronuclear H-1-C-13 HSQC spectroscopy, which did not detect resonances for the C-13(5)-H, C-13(10)-H, and C-13(15)-H hydrogen atoms whereas strong signals were detected for the C-13-labeled chromopeptide. The most pronounced C-13-chemical shift difference between chromopeptide and intact receptor domain was that of the C-13(4)-resonance, which could be consistent with an increased conformational energy of the C-4-C-5-C-6-N-2 dihedral angle in the intact protein in the Pr state. Nuclear Overhauser effect spectroscopy experiments of the C-13-labeled chromopeptide, where chromophore-protein interactions are expected to be reduced, were consistent with a ZZZssa conformation, which has also been found for the biliverdin chromophore in the x-ray structure of a fragment of Deinococcus radiodurans bacteriophytochrome in the Pr form.