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 P_fr, 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-R_C and P_fr. In the biliverdin adduct, an additional component occurs that probably reflects a transition between two Meta-R_C 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.Phytochromes are red-light photoreceptors occurring in plants, bacteria, and fungi where they control important developmental processes (1–6). The discovery of microbial phytochromes from genome sequencing (7–9) provided new prospects for biochemical, spectroscopic and structural analyses of this light sensor family. Agp1 (AtBphP1)³ from the soil bacterium Agrobacterium tumefaciens is a typical member of the widespread family of proteobacterial phytochromes (10, 11) and is the subject of the present study.The domain arrangement of canonical phytochromes consists of an N-terminal photosensory domain, including PAS, GAF, and PHY domains and a C-terminal regulatory kinase domain (see, e.g. Ref. 3). Bacterial phytochromes lack the N-terminal extension, and the PAS module insertion of plant phytochromes (3). In most of the bacterial phytochromes, the C-terminal regulatory domain is a histidine kinase (4). These kinases form homodimers as functional units (12) where the subunits transphosphorylate each other (13). The cofactors are linear tetrapyrroles that are covalently attached via a thioether linkage (14) to the side chains of specific conserved cysteine residues. The native chromophore of plant phytochromes is phytochromobilin (PΦB) (14), some cyanobacterial phytochromes incorporate phycocyanobilin (PCB) (15, 16), and all other bacterial phytochromes bind biliverdin (BV) (10, 11). Whereas the chromophore binding site of the more reduced bilins PΦB and PCB is located in the GAF domain, the binding site of BV is close to the N terminus upstream of the PAS domain (4, 11). The two distinct binding sites apparently require a specific substituent at the C3 carbon of pyrrole ring A, either an ethylidene (PΦB and PCB) or a vinyl (BV) group, for covalent attachment of the bilin chromophore (4). The holophytochrome assembly that includes covalent attachment of the chromophore is an autocatalytic process implying an intrinsic bilin C-S lyase activity of the apophytochrome (17). Kinetic studies of the autoassembly in vitro showed that ligation of the chromophore is the ultimate step following incorporation in the binding pocket and internal protonation (18).Phytochromes display photochromicity involving two either thermally stable or long-lived states, P_r and P_fr (red and far-red absorbing forms), that can be reversibly converted by light of appropriate wavelengths. The P_r to P_fr photoconversion is initiated by a rapid Z/E isomerization of the C-D methine bridge of the bilin chromophore (19–22) leading within picoseconds to the formation of the Lumi-R intermediate (23, 24). The following thermal relaxations via Meta-R_A and Meta-R_C intermediates to P_fr proceed on the time scale of microseconds and milliseconds (25–28).Assembly of Agp1 with locked BV derivatives showed that the geometry of the C-D methine bridge is 15Z_anti in P_r and 15E_anti in P_fr (29) suggesting that this methine bridge remains in the anti conformation during photoconversion. The crystal structures of the chromophore binding domains of the bacteriophytochromes from Deinococcus radiodurans and Rhodopseudomonas palustris revealed that the BV chromophore adopts a 5Z_syn,10Z_syn,15Z_anti configuration/conformation in the P_r state (30–32). The 5Z_syn geometry of the A-B methine bridge in the P_r state was confirmed by assembly of Agp1 with the corresponding locked BV chromophore (33). Recently, heteronuclear NMR investigations and crystallographic studies on the complete photosensory domain of the cyanobacterial phytochrome Cph1 from Synechocystis showed that the PCB chromophore is also in the 5Z_syn,10Z_syn,15Z_anti geometry in P_r (34, 35).Because the locked 5Z_syn adduct of Agp1 did not show a P_fr-like photo-product, conformational changes of the A-B methine bridge in the thermal relaxation cascade have been predicted (33). Flash photolysis experiments with this adduct suggested that these changes occur in the Meta-R_A to Meta-R_C transition (36). The stereochemistry of the A-B methine bridge in the P_fr state and in the preceding intermediates could not be determined unambiguously yet. Recent studies with doubly locked chromophores suggest that the C5–C6 single bond undergoes a thermal rotation from syn to anti in the photoconversion of Agp1, whereas an additional Z/E isomerization around the C4C5 double bond (hula-twist mechanism) was postulated for Agp2 (37). However, the crystal structure of the photosensory domain of the bacteriophytochrome PaBphP in its P_fr-enriched dark-adapted state favors the 5Z_syn conformation of the BV chromophore (38). Structural changes of the A-B methine bridge were excluded for the PCB chromophore of Cph1 on the basis of heteronuclear NMR (34), whereas low temperature Fourier transform IR studies on plant phytochrome suggested an environmental change of the ring A carbonyl group and/or a twist of the A-B methine bridge (39).The mechanism by which the signal is transmitted from the bilin chromophore to the protein is still obscure. The recent three-dimensional structures of the complete photosensory domains of Cph1 (35) and PaBphP (38) reveal key interactions between GAF and PHY domains in the corresponding dark states reflecting P_r and P_fr, respectively. In view of the intrinsic differences between the two phytochromes, it is not trivial to differentiate which of the numerous structural differences arise from light-induced conformational changes and are thus potentially important for signal transmission. We note that many approaches to provide a clue on the mechanism of signal transmission from the bilin chromophore to its proximate environment imply that this process is exclusively coupled to the photo-isomerization localized at ring D and its environment and that the chromophore then remains a passive element in the thermal relaxation cascade. This point of view is supported by recent results from femtosecond stimulated Raman spectroscopy suggesting that the chromophore structures in Lumi-R and P_fr are very similar (24). On the other hand, size exclusion chromatography experiments demonstrated that the global conformational changes observed for the P_fr state of Agp1 WT are absent in constructs (locked 5Zs adduct and mutants D197A and H250A), where the formation of P_fr is inhibited but the primary photoreaction proceeds (33, 40). These results are difficult to explain in terms of an ultra-fast signal transmission from the chromophore to the surrounding residues in its pocket.Light-induced conformational changes at the surface of plant phytochrome were observed by using covalently attached labels that are sensitive to the polarity of the microenvironment (41, 42). Due to the accessibility of several binding sites (i.e. the sulfhydryl groups of cysteines) in these experiments, the labeling was unspecific preventing further assignment of the observed changes to particular regions of the protein. Time-resolved absorption measurements with a covalently attached fluorescein derivative showed that the changes occur in the Meta-R_C to P_fr transition (41). In the present work with Agp1 phytochrome, we take advantage of the highly reactive sulfhydryl group of Cys-20, the genuine binding site of the BV chromophore, to specifically attach a fluorescein derivative. We observed that this construct assembles with PCB and BV forming noncovalent photochromic adducts, spectrally and kinetically undisturbed by the fluorescein label. Upon photo-conversion, the absorption band of the label displays a bathochromic shift and increase in ellipticity suggesting that the label moves in a more hydrophobic and asymmetric environment in the P_fr state. The label thus serves as a polarity probe at the entrance of the binding pocket. We postulate that these polarity changes reflect conformational changes of the A-B methine of the bilin chromophore and/or the microenvironment of ring A at the entrance of the binding pocket. Time-resolved measurements reveal that the changes occur in the Meta-R_A to Meta-R_C and Meta-R_C to P_fr transitions. Analogous results were obtained with the V249C mutant of Agp1 in which both the fluorescein probe and the PCB chromophore are covalently attached.