CHROMOPHORE STRUCTURE IN THE PHOTOCYCLE OF THE CYANOBACTERIAL PHYTOCHROME CPH1

¹ University of Oxford

^* To whom correspondence should be addressed. E-mail: jasper{at}biop.ox.ac.uk.

Submitted on March 1, 2006
Revised on April 21, 2006
Accepted on 17 May 2006

	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 C₄ 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 µs and ms 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 photoisomerisation 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 labelled 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 ¹³C labelled 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. NOESY experiments of the ¹³C labelled 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 D. radiodurans bacteriophytochrome in the Pr form (1).

Key Words: Cph1, GAIO calculation, NMR spectroscopy, Photoisomerisation, Phytochrome, TDDFT

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