Berthold Borucki

Light-induced Proton Release of Phytochrome Is Coupled to the Transient Deprotonation of the Tetrapyrrole Chromophore

The Pr → Pfr phototransformation of the bacteriophytochrome Agp1 from Agrobacterium tumefaciens and the structures of the biliverdin chromophore in the parent states and the cryogenically trapped intermediate Meta-RC were investigated with resonance Raman spectroscopy and flash photolysis. Strong similarities with the resonance Raman spectra of plant phytochrome A indicate that in Agp1 the methine bridge isomerization state of the chromophore is ZZZasa in Pr and ZZEssa in Pfr, with all pyrrole nitrogens being protonated. Photoexcitation of Pr is followed by (at least) three thermal relaxation components in the formation of Pfr with time constants of 230 μs and 3.1 and 260 ms. H2O/D2O exchange reveals kinetic isotope effects of 1.9, 2.6, and 1.3 for the respective transitions that are accompanied by changes of the amplitudes. The second and the third relaxation correspond to the formation and decay of Meta-RC, respectively. Resonance Raman measurements of Meta-RC indicate that the chromophore adopts a deprotonated ZZE configuration. Measurements with a pH indicator dye show that formation and decay of Meta-RC are associated with proton release and uptake, respectively. The stoichiometry of the proton release corresponds to one proton per photoconverted molecule. The coupling of transient chromophore deprotonation and proton release, which is likely to be an essential element in the Pr → Pfr photocon-version mechanism of phytochromes in general, may play a crucial role for the structural changes in the final step of the Pfr formation that switch between the active and the inactive state of the photoreceptor.

Highly conserved residues Asp-197 and His-250 in agp1 phytochrome control the proton affinity of the chromophore and Pfr formation

The mutants H250A and D197A of Agp1 phytochrome from Agrobacterium tumefaciens were prepared and investigated by different spectroscopic and biochemical methods. Asp-197 and His-250 are highly conserved amino acids and are part of the hydrogen-bonding network that involves the chromophore. Both substitutions cause a destabilization of the protonated chromophore in the Pr state as revealed by resonance Raman and UV-visible absorption spectroscopy. Titration experiments demonstrate a lowering of the pKa from 11.1 (wild type) to 8.8 in H250A and 7.2 in D197A. Photoconversion of the mutants does not lead to the Pfr state. H250A is arrested in a meta-Rc-like state in which the chromophore is deprotonated. For H250A and the wild-type protein, deprotonation of the chromophore in meta-Rc is coupled to the release of a proton to the external medium, whereas the subsequent proton re-uptake, linked to the formation of the Pfr state in the wild-type protein, is not observed for H250A. No transient proton exchange with the external medium occurs in D197A, suggesting that Asp-197 may be the proton release group. Both mutants do not undergo the photo-induced protein structural changes that in the wild-type protein are detectable by size exclusion chromatography. These conformational changes are, therefore, attributed to the meta-Rc → Pfr transition and most likely coupled to the transient proton re-uptake. The present results demonstrate that Asp-197 and His-250 are essential for stabilizing the protonated chromophore structure in the parent Pr state, which is required for the primary photochemical process, and for the complete photo-induced conversion to the Pfr state.

Light-Induced Conformational Changes of the Chromophore and the Protein in Phytochromes: Bacterial Phytochromes as Model Systems

Recombinant phytochromes Agp1 and Agp2 from Agrobacterium tumefaciens are used as model phytochromes for biochemical and biophysical studies. In biliverdin binding phytochromes the site for covalent attachment of the chromophore lies in the N-terminal region of the protein, different from plant phytochromes. The issue which stereochemistry the chromophore adopts in the so-called Pr and Pfr forms is addressed by using a series of locked chromophores which form spectrally characteristic adducts with Agp1 and Agp2. Studies on light-induced conformational changes of Agp1 give an insight into how the intrinsic histidine kinase is modulated by light. Comparison of the crystal structure of an Agp1 fragment with other phytochrome crystal structures supports the idea that a light induced rearrangement of subunits within the homodimer modulates the activity of the kinase.

Locked 5Zs-biliverdin blocks the Meta-RA to Meta-RC transition in the functional cycle of bacteriophytochrome Agp1

The bacteriophytochrome Agp1 was reconstituted with a locked 5Zs‐biliverdin in which the C4 C5 and C5–C6 bonds of the methine bridge between rings A and B are fixed in the Z and syn configuration/conformation, respectively. In Agp1‐5Zs the photoconversion proceeds via the Lumi‐R intermediate to Meta‐RA, but the following millisecond‐transition to Meta‐RC is blocked. Consistently, no transient proton release was detected. The photoconversion of Agp1‐5Zs is apparently arrested in a Meta‐RA‐like intermediate, since the subsequent syn to anti rotation around the C5–C6 bond is prevented by the lock. The Meta‐RA‐like photoproduct was characterized by its distinctive CD spectrum suggesting a reorientation of ring D.

Chromophore reorientation during the photocycle of bacteriorhodopsin: experimental methods and functional significance

Light-induced isomerization leads to orientational changes of the retinylidene chromophore of bacteriorhodopsin in its binding pocket. The chromophore reorientation has been characterized by the following methods: polarized absorption spectroscopy in the visible, UV and IR; polarized resonance Raman scattering; solid-state deuterium nuclear magnetic resonance; neutron and X-ray diffraction. Most of these experiments were performed at low temperatures with bacteriorhodopsin trapped in one or a mixture of intermediates. Time-resolved measurements at room temperature with bacteriorhodopsin in aqueous suspension can currently only be carried out with transient polarized absorption spectroscopy in the visible. The results obtained to date for the initial state and the K, L and M intermediates are presented and discussed. The most extensive data are available for the M intermediate, which plays an essential role in the function of bacteriorhodopsin. For this intermediate the various methods lead to a consistent picture: the curved all-trans polyene chain in the initial state straightens out in the M intermediate (13-cis) and the chain segment between C(5) and C(13) tilts upwards in the direction of the cytoplasmic surface. The kink at C(13) allows the positions of beta-ionone ring and Schiff base nitrogen to remain approximately fixed.

A Polarity Probe for Monitoring Light-induced Structural Changes at the Entrance of the Chromophore Pocket in a Bacterial Phytochrome

Light-induced structural changes at the entrance of the chromophore pocket of Agp1 phytochrome were investigated by using a thiol-reactive fluorescein derivative that is covalently attached to the genuine chromophore binding site (Cys-20) and serves as a polarity probe. In the apoprotein, the absorption spectrum of bound fluorescein is red-shifted with respect to that of the free label suggesting that the probe enters the hydrophobic chromophore pocket. Assembly of this construct with the chromophores phycocyanobilin or biliverdin is associated with a blue-shift of the fluorescein absorption band indicating the displacement of the probe out of the pocket. The probe does not affect the photochromic and kinetic properties of the noncovalent bilin adducts. Upon photoconversion to Pfr, the probe spectrum undergoes again a bathochromic shift and a strong rise in CD indicating a more hydrophobic and asymmetric environment. We propose that the environmental changes of the probe reflect conformational changes at the entrance of the chromophore pocket and are indicative for rearrangements of the chromophore ring A. Flash photolysis measurements showed that the absorption changes of the probe are kinetically coupled to the formation of Meta-RC and Pfr. In the biliverdin adduct, an additional component occurs that probably reflects a transition between two Meta-RC substates. Analogous results to that of the noncovalent phycocyanobilin adduct were obtained with the mutant V249C in which probe and chromophore are covalently attached. The conformational changes of the chromophore are correlated to proton transfer to the protein surface.

Proton transfer in the photoreceptors phytochrome and photoactive yellow protein

Light-induced activation of the photoreceptors phytochrome and photoactive yellow protein (PYP) is accompanied by protonation changes of the respective chromophores and key residues in the protein moiety. For both systems, proton exchange with the external medium could be observed with pH electrode measurements and with UV-visible absorption spectroscopy using appropriate pH indicator dyes. From these signals, the stoichiometry of proton release and uptake, respectively, was determined by different calibration procedures which will be presented and discussed. Kinetic information on these processes is only available from time-resolved measurements with pH indicator dyes. Vibrational spectroscopy methods such as Fourier transform infrared spectroscopy and resonance Raman scattering provided information on the protonation state of individual functional groups suggesting that internal proton transfer processes are involved as well. Deuterium kinetic isotope effects that occurred in the Pr --> Pfr phototransformation of the bacteriophytochromes Cph1 and Agp1 were consistent with proton transfer reactions as rate-limiting steps. In contrast, the apparent rate constants in the photocycle of PYP exhibited only small kinetic isotope effects that could not be interpreted conclusively. Possible mechanisms of proton transfer in the activation of phytochrome and PYP will be discussed.

Regulation of the Ppr Histidine Kinase by Light-Induced Interactions between Its Photoactive Yellow Protein and Bacteriophytochrome Domains

Ppr is a unique bacteriophytochrome that bleaches rather than forming a far-red-shifted Pfr state upon red light activation. Ppr is also unusual in that it has a blue light photoreceptor domain, PYP, which is N-terminally fused to the bacteriophytochrome domain (Bph). When both photoreceptors are activated by light, the fast phase of Bph recovery (1 min lifetime) corresponds to the formation of an intramolecular long-lived complex between the activated PYP domain and the Bph domain (lifetime of 2-3 days). Since this state is unusually long-lived as compared to other intermediates in the photocycle of both PYP and Bph, we interpret this as formation of a metastable complex between activated PYP and Bph domains that takes days to relax. In the metastable complex, the PYP domain is locked in its activated UV absorbing state and the Bph domain is in a slightly red-shifted state (from 701 to 702 nm), which is photochemically inactive to red or white light. The amount of metastable complex formed increases with the degree of prior activation of PYP, reaching a maximum of 50% when PYP is fully activated compared to 0% when no PYP is activated. The saturation of complex formation at 50% is believed to be due to light-induced heterogeneity within the Ppr dimer. UV irradiation (365 nm) of the metastable complex state photoreverses the activated PYP and the red-shifted Bph to the initial dark state within seconds. We therefore postulate that Ppr functions as a UV-red light sensor and describe the different Ppr states that can be obtained depending on the light quality. Both red and white light upregulate the autokinase activity, while it is downregulated in the dark. The physiological state of Ppr is most likely a mixture of three different states, dark, metastable complex, and red light-activated, with fractional populations whose amounts depend on the light quality of the environment and that regulate the extent of phosphorylation by the kinase.