Kinetische Untersuchungen zur Bildung des aktivierten Zustandes in den bakteriellen Phytochromen Cph1 und Agp1
Final Report Abstract
Phytochromes are red-light photoreceptors occurring in plants, bacteria and fungi where they control important developmental processes. Phytochromes display photochromic behaviour involving two almost thermostable states, Pr and Pfr, with distinct absorption spectra that can be reversibly interconverted by red and far-red light. The bacterial phytochromes Cph1 and Agp1 are light-regulated histidine kinases. The regulation is mediated by a bilin cofactor that assembles autocatalytically with the apoprotein. The native cofactors of Cph1 and Agp1 are phycocyanobilin (PCB) and biliverdin (BV), respectively. The activity of the holoprotein is then modulated by light of appropriate wavelength. The kinetics of these two processes – assembly and photoconversion – were investigated by timeresolved optical spectroscopy. From stopped-flow mixing experiments with milliseconds time resolution a three-step model for the self-assembly mechanism was developed. In a first unresolved step, a non-covalent complex of the apoprotein and the bilin chromophore in a cyclic configuration is formed. The second step with a time constant of 100 – 150 ms is associated with a major red-shift and an increase of the Q-band absorption indicative of protonation and formation of a more extended conformation of the chromophore. Since the pH-dependence of this component differs in the assemblies of Cph1 and Agp1 with their native chromophores, the mechanisms of chromophore protonation are supposed to be different in both phytochromes. The third step reflecting the formation of the covalent bond occurs in the time range of seconds in Cph1 and is slower by a factor of ~10 in Agp1. The kinetics of the Pr to Pfr photoconversion and proton transfer to the aqueous medium were investigated by flash photolysis. While the three transitions in Agp1-BV are spectrally very similar to that of plant phytochrome, the spectral characteristic of the Pr to Pfr photoconversion differs significantly in Cph1-PCB. The kinetics and the patterns of proton transfer are very similar in both bacterial phytochromes, though. Transient proton release and partial reuptake were correlated with the formation of the Meta-RC intermediate and Pfr, respectively. In Agp1, proton release is coupled to transient deprotonation of the BV chromophore that may play a crucial role in the activation of the photoreceptor. Mutational studies showed that Asp-197 and His-250 are essential for stabilizing the protonated chromophore structure in the Pr state, which is required for the primary photochemical process, and for the complete conversion to the Pfr state. Stationary circular dichroism spectroscopy and flash photolysis with covalent and noncovalent bilin adducts of Agp1 demonstrated that the covalent attachment of the chromophore is not required for functional integrity of Agp1. The Pr and Pfr states of the covalent PCB adduct of Agp1 are spectrally similar to that of Cph1 while the spectral characteristic of the photoconversion of Agp1-PCB resembles that of Agp1-BV. Proton release also occurs in Agp1-PCB but the proton reuptake component is absent. The same kinetics of protonation changes was observed when a pH-indicator dye is covalently attached to the protein. Flash photolysis with the locked 5Zs-BV adduct of Agp1 showed that the Meta-RA to Meta-RC transition is blocked suggesting that a syn to anti rotation of the A-B methine bridge occurs in the respective transition in the unlocked adduct. In the 18Et-BV adduct of Agp1 the formation of Pfr is accelerated by more than a factor of 10. This observation indicates that the pyrrole ring D is not only involved in the rapid photoisomerization but also in the much slower relaxations. Flash photolysis with a deletion mutant lacking the PHY domain reveals that the reaction pathway of the photoconversion from the dark state to the Meta-RC-like photoproduct is completely different from the respective transitions in wild type Agp1. The PHY domain thus controls the conformational changes of the chromophore during the relaxation after photoexcitation.
Publications
- (2005) Lightinduced proton release of phytochrome is coupled to the transient deprotonation of the tetrapyrrole chromophore. J. Biol. Chem. 280, 34358-34364
B. Borucki, D. von Stetten, S. Seibeck, T. Lamparter, N. Michael, M. A. Mroginski, H. Otto, D. H. Murgida, M. P. Heyn, and P. Hildebrandt
- (2006) Proton transfer in the photoreceptors phytochrome and Photoactive Yellow Protein. Photochem. Photobiol. Sci. 5, 553 - 566
B. Borucki
- (2006) Secondary structural changes in the autoassembly and photoconversion of bacteriophytochromes. BESSY Annual Report 2005, 434-435
B. Borucki, H. Otto, S. Seibeck, M. P. Heyn and T. Lamparter
- (2007) Highly conserved residues Asp-197 and His-250 in Agp1 phytochrome control the proton affinity of the chromophore and Pfr formation. J. Biol. Chem. 282, 2116–2123
D. von Stetten, S. Seibeck, N. Michael, P. Scheerer, M. A. Mroginski, D. H. Murgida, N. Krauß, M. P. Heyn, P. Hildebrandt, B. Borucki, and T. Lamparter
- (2007) Locked 5Zs-biliverdin blocks the Meta-RA to Meta-RC transition in the functional cycle of bacteriophytochrome Agp1. FEBS Lett. 581, 5425–5429
S. Seibeck, B. Borucki, H. Otto, K. Inomata, H. Khawn, H. Kinoshita, N. Michael, T. Lamparter, and M. P. Heyn
