Chromophore structure in the photocycle of the cyanobacterial phytochrome Cph1

Abstract

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 13C isotope substitutions in the chromophore. 13C-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 C15 and C5, which were much less pronounced. Ab initio calculations of the isotropic shielding and optical transition energies identify a region for C4-C5-C6-N2 dihedral angle changes where deshielding of C4 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 C5 could satisfy the observed NMR and visible absorption changes. Alternatively, C15 Z-E photoisomerization, although expected to lead to a small change of the chemical shift of C15, in addition to changes of the C4-C5-C6-N2 dihedral angle could be consistent with visible absorption changes and the chemical shift difference at C4. NMR spectroscopy of a 13C-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 1H-13C HSQC spectroscopy, which did not detect resonances for the 13C5-H, 13C10-H, and 13C15-H hydrogen atoms whereas strong signals were detected for the (13)C-labeled chromopeptide. The most pronounced 13C-chemical shift difference between chromopeptide and intact receptor domain was that of the 13C4-resonance, which could be consistent with an increased conformational energy of the C4-C5-C6-N2 dihedral angle in the intact protein in the Pr state. Nuclear Overhauser effect spectroscopy experiments of the 13C-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.

FIGURE 1

¹H-broadband decoupled ¹³C-NMR spectra of labeled (solid lines) and unlabeled (dashed lines) intact Cph1-N515 (A) and chromopeptide (B) identify signals belonging to ¹³C₄, ¹³C₅, ¹³C₉, ¹³C₁₀, ¹³C₁₁, ¹³C₁₅, and ¹³C_19. Labeled and unlabeled Cph1-N515 and derived chromopeptides were at 200-μM concentration, and spectra shown for comparison are not scaled in intensity.

FIGURE 2

Conformational exchange reactions of the chromophore in the intact protein. (A) ¹³C-NMR spectra of the ¹³C₅ and ¹³C₁₅ carbons in Cph1-N515 (solid line) and chromopeptide (dashed line) under identical experimental conditions and concentration. Arrows indicate chromophore peaks. (B) ¹H-¹³C HSQC crosspeaks for the ¹³C₅-H and ¹³C₁₅-H protons in the labeled chromopeptide. No peaks were observed in this region in unlabeled chromopeptide under identical conditions. (C) ¹³C-NMR spectra of the ¹³C₁₀ triplet in Cph1-N515 (solid line) and chromopeptide (dashed line), indicated with arrows. (D). ¹H-¹³C HSQC crosspeaks of the ¹³C₁₀-H triplet in the labeled chromopeptide (light contours) shown together with the unlabeled chromopeptide, which shows contributions from superimposed resonances at natural abundance in this region (dark contours).

FIGURE 3

¹H-¹H-NOESY traces parallel to F2 at 5.68 and 6.29 ppm of the ¹³C-labeled chromopeptide. NOESY experiment was run without ¹³C-decoupling in F2 to demonstrate the J_CH coupling in the C₅-H and C₁₅-H bonds. The brackets and arrows indicate the position of collapsed diagonal peaks as seen in fully decoupled experiments. Artifacts are marked with “X”, and NOE crosspeaks are marked with small arrows.

FIGURE 4

¹³C-NMR spectroscopy of Pr and Pfr states. The Pr state was obtained in pure form after saturating illumination with >705 nm far red light. A mixture of Pfr and Pr states was obtained after illumination of the concentrated sample in a thin capillary with red light, 640 nm.

FIGURE 5

Molecular properties with C₄-C₅-C₆-N₂ dihedral angle changes. A relaxed C₄-C₅-C₆-N₂ dihedral angle scan was performed of the ZZZ(s)sa (A–C) (•) and EZZ(s)sa (D–F) (▴) geometries. DFT conformational energies (/kJ/mol) (A and D) are given relative to the lowest conformation. Isotropic chemical shielding values are given for the ¹³C₄-carbon relative to TMS (/ppm) (B and E). TDDFT optical transition energies computed for the pure HOMO-LUMO transition (/eV) (C and F).

FIGURE 6

Chromophore models and key calculated properties. The conformational models shown are the optimized geometries at the DFT MPW1PW91 6-31G(d,p) level also used for the GIAO and TDDFT calculations but additionally contain the carboxylic acid groups which were excluded in the calculations. Dihedral angle restraints used in the geometry optimization, the calculated isotropic shielding for C₄ and the TDDFT excitation energy are listed as a summary of the results, which are discussed. (A) ZZZssa model obtained after geometry optimization as taken from the Bph x-ray structure. (B) ZZEssa structure based on A. (C) ZZZssa structure including only the C₄-C₅-C₆-N₂ dihedral angle restraint. (D) EZZasa structure based on C.