Biliverdin Binds Covalently to Agrobacterium Phytochrome Agp1 via Its Ring A Vinyl Side Chain

Abstract

The widely distributed phytochrome photoreceptors carry a bilin chromophore, which is covalently attached to the protein during a lyase reaction. In plant phytochromes, the natural chromophore is coupled by a thioether bond between its ring A ethylidene side chain and a conserved cysteine residue within the so-called GAF domain of the protein. Many bacterial phytochromes carry biliverdin as natural chromophore, which is coupled in a different manner to the protein. In phytochrome Agp1 of Agrobacterium tumefaciens, biliverdin is covalently attached to a cysteine residue close to the N terminus (position 20). By testing different natural and synthetic biliverdin derivatives, it was found that the ring A vinyl side chain is used for chromophore attachment. Only those bilins that have ring A vinyl side chain were covalently attached, whereas bilins with an ethylidene or ethyl side chain were bound in a noncovalent manner. Phycocyanobilin, which belongs to the latter group, was however covalently attached to a mutant in which a cysteine was introduced into the GAF domain of Agp1 (position 249). It is proposed that the regions around positions 20 and 249 are in close contact and contribute both to the chromophore pocket. In competition experiments it was found that phycocyanobilin and biliverdin bind with similar strength to the wild type protein. However, in the V249C mutant, phycocyanobilin bound much more strongly than biliverdin. This finding could explain why during phytochrome evolution in cyanobacteria, the chromophore-binding site swapped from the N terminus into the GAF domain. The widely distributed phytochrome photoreceptors carry a bilin chromophore, which is covalently attached to the protein during a lyase reaction. In plant phytochromes, the natural chromophore is coupled by a thioether bond between its ring A ethylidene side chain and a conserved cysteine residue within the so-called GAF domain of the protein. Many bacterial phytochromes carry biliverdin as natural chromophore, which is coupled in a different manner to the protein. In phytochrome Agp1 of Agrobacterium tumefaciens, biliverdin is covalently attached to a cysteine residue close to the N terminus (position 20). By testing different natural and synthetic biliverdin derivatives, it was found that the ring A vinyl side chain is used for chromophore attachment. Only those bilins that have ring A vinyl side chain were covalently attached, whereas bilins with an ethylidene or ethyl side chain were bound in a noncovalent manner. Phycocyanobilin, which belongs to the latter group, was however covalently attached to a mutant in which a cysteine was introduced into the GAF domain of Agp1 (position 249). It is proposed that the regions around positions 20 and 249 are in close contact and contribute both to the chromophore pocket. In competition experiments it was found that phycocyanobilin and biliverdin bind with similar strength to the wild type protein. However, in the V249C mutant, phycocyanobilin bound much more strongly than biliverdin. This finding could explain why during phytochrome evolution in cyanobacteria, the chromophore-binding site swapped from the N terminus into the GAF domain. Phytochromes are biliprotein photoreceptors that were discovered in plants (1Butler W.L. Norris K.H. Siegelman H.W. Hendricks S.B. Proc. Natl. Acad. Sci. U. S. A. 1959; 45: 1703-1708Crossref PubMed Google Scholar) but were recently also found in bacteria (2Lamparter T. Mittmann F. Gärtner W. Börner T. Hartmann E. Hughes J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11792-11797Crossref PubMed Scopus (157) Google Scholar, 3Yeh K.C. Wu S.H. Murphy J.T. Lagarias J.C. Science. 1997; 277: 1505-1508Crossref PubMed Scopus (451) Google Scholar, 4Davis S.J. Vener A.V. Vierstra R.D. Science. 1999; 286: 2517-2520Crossref PubMed Scopus (296) Google Scholar), fungi (5Bhoo S.H. Davis S.J. Walker J. Karniol B. Vierstra R.D. Nature. 2001; 414: 776-779Crossref PubMed Scopus (253) Google Scholar), and slime molds (6Starostzik C. Marwan W. FEBS Lett. 1995; 370: 146-148Crossref PubMed Scopus (46) Google Scholar). The photocycle of phytochromes has two long-lived forms, the red-absorbing form (Pr), 1The abbreviations used are: Pr, red-absorbing form of phytochrome; Pfr, far red-absorbing form of phytochrome; BV, biliverdin; PCB, phycocyanobilin; PΦB, phytochromobilin. which is synthesized in darkness, and the far red-absorbing form (Pfr). Both forms are reversibly interconverted by light. In general, both forms are stable in darkness, but exceptions are often found. The Pfr form of many plant phytochromes (7Mancinelli A. Kendrick R.E. Kronenberg G.H.M. Photomorphogenesis of Plants. 2nd Ed. Kluwer Academic Publishers, Dordrecht, The Netherlands1994: 211-269Google Scholar) and phytochrome 1 from Agrobacterium tumefaciens (here termed Agp1) (8Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (164) Google Scholar) slowly converts to Pr in darkness. In the case of Bradyrhizobium phytochrome and Agrobacterium phytochrome 2, Pr converts to Pfr in darkness (9Giraud E. Fardoux J. Fourrier N. Hannibal L. Genty B. Bouyer P. Dreyfus B. Vermeglio A. Nature. 2002; 417: 202-205Crossref PubMed Scopus (172) Google Scholar, 10Karniol B. Vierstra R.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2807-2812Crossref PubMed Scopus (131) Google Scholar). Typically, the phytochrome proteins consist of an N-terminal chromophore module, to which one bilin chromophore is covalently attached, and a C-terminal output module. In prokaryotic phytochromes, the output module is most often a histidine kinase (2Lamparter T. Mittmann F. Gärtner W. Börner T. Hartmann E. Hughes J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11792-11797Crossref PubMed Scopus (157) Google Scholar, 8Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (164) Google Scholar), but other C-terminal modules are also possible (9Giraud E. Fardoux J. Fourrier N. Hannibal L. Genty B. Bouyer P. Dreyfus B. Vermeglio A. Nature. 2002; 417: 202-205Crossref PubMed Scopus (172) Google Scholar, 10Karniol B. Vierstra R.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2807-2812Crossref PubMed Scopus (131) Google Scholar). Almost all plant phytochromes have a histidine-kinase like domain and an additional ∼300-amino acid stretch with two PAS domains, which is located between the chromophore module and the histidine-kinase-like domain (11Montgomery B.L. Lagarias J.C. Trends Plant Sci. 2002; 7: 357-366Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Within the chromophore module, the GAF domain and a PHY domain, which is located C-terminal of the GAF domain, can be distinguished. The GAF domain is the region most homologous among the phytochrome family. Further N-terminal of this domain, there are 140–200 additional amino acids of lower homology. Within this region, a PAS domain was identified in some phytochrome species (11Montgomery B.L. Lagarias J.C. Trends Plant Sci. 2002; 7: 357-366Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). The domain structure of Agrobacterium phytochrome Agp1 is given in Fig. 1. Plant phytochromes carry either phytochromobilin (PΦB) or phycocyanobilin (PCB) as chromophore (12Rüdiger W. Thümmler F. Kendrick R.E. Kronenberg G.H.M. Photomorphogenesis of Plants. 2nd Ed. Kluwer Academic Publishers, Dordrecht, The Netherlands1994: 51-69Google Scholar, 13Wu S.H. McDowell M.T. Lagarias J.C. J. Biol. Chem. 1997; 272: 25700-25705Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Both bilins are synthesized from biliverdin (BV) by reduction of the BV ring A double bond and a concomitant conversion of ring A vinyl to ethylidene. For PCB biosynthesis, the ring D vinyl group is further reduced to an ethyl group (14Kohchi T. Mukougawa K. Frankenberg N. Masuda M. Yokota A. Lagarias J.C. Plant Cell. 2001; 13: 425-436Crossref PubMed Scopus (203) Google Scholar, 15Frankenberg N. Mukougawa K. Kohchi T. Lagarias J.C. Plant Cell. 2001; 13: 965-978Crossref PubMed Scopus (195) Google Scholar). In plant phytochromes, the chromophore binds covalently to the protein by a thioether bond between the ring A ethylidene side chain of the chromophore (12Rüdiger W. Thümmler F. Kendrick R.E. Kronenberg G.H.M. Photomorphogenesis of Plants. 2nd Ed. Kluwer Academic Publishers, Dordrecht, The Netherlands1994: 51-69Google Scholar) and a conserved cysteine within the GAF domain. In cyanobacteria, two types of phytochromes are distinguished with respect to chromophore attachment. In some phytochromes such as Cph1 from Synechocystis or CphA of Calothrix, the cysteine residue is conserved (2Lamparter T. Mittmann F. Gärtner W. Börner T. Hartmann E. Hughes J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11792-11797Crossref PubMed Scopus (157) Google Scholar, 16Jorissen H.J. Quest B. Remberg A. Coursin T. Braslavsky S.E. Schaffner K. Tandeau de Marsac N. Gärtner W. Eur. J. Biochem. 2002; 269: 2662-2671Crossref PubMed Scopus (54) Google Scholar). These phytochromes also attach PCB or PΦB in a covalent manner. Most cyanobacteria synthesize large amounts of PCB as phycobiliprotein compound, and PCB was found as a natural chromophore of Cph1 (17Hübschmann T. Börner T. Hartmann E. Lamparter T. Eur. J. Biochem. 2001; 268: 2055-2063Crossref PubMed Scopus (83) Google Scholar). Other cyanobacterial phytochromes, such as CphB from Calothrix, are missing the chromophore-binding cysteine in the GAF domain. Recombinant CphB incorporates PCB to a photoactive adduct, but the attachment is noncovalent, and the bilin can easily be removed from the protein. When a cysteine was introduced at the appropriate site of CphB, the protein bound PCB covalently (16Jorissen H.J. Quest B. Remberg A. Coursin T. Braslavsky S.E. Schaffner K. Tandeau de Marsac N. Gärtner W. Eur. J. Biochem. 2002; 269: 2662-2671Crossref PubMed Scopus (54) Google Scholar). Phytochromes of other bacteria are all missing the chromophore-binding cysteine in the GAF domain (4Davis S.J. Vener A.V. Vierstra R.D. Science. 1999; 286: 2517-2520Crossref PubMed Scopus (296) Google Scholar, 5Bhoo S.H. Davis S.J. Walker J. Karniol B. Vierstra R.D. Nature. 2001; 414: 776-779Crossref PubMed Scopus (253) Google Scholar, 8Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (164) Google Scholar). As far as analyzed, these phytochromes incorporate BV as natural chromophore (5Bhoo S.H. Davis S.J. Walker J. Karniol B. Vierstra R.D. Nature. 2001; 414: 776-779Crossref PubMed Scopus (253) Google Scholar, 8Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (164) Google Scholar), but recombinant proteins assemble to photoactive adducts also with PCB and PΦB (4Davis S.J. Vener A.V. Vierstra R.D. Science. 1999; 286: 2517-2520Crossref PubMed Scopus (296) Google Scholar, 8Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (164) Google Scholar). For phytochrome Agp1 from A. tumefaciens, it was shown by site-directed mutagenesis that BV is covalently bound to a cysteine residue close to the N terminus of the protein (8Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (164) Google Scholar). These results have been confirmed by mass spectrometry analyses. 2M. Carrascal, J. Abian, N. Michael, G. Rottwinkler, and T. Lamparter, personal communication. This cysteine residue is highly conserved within the group of BV-binding phytochromes and is probably also used as attachment site in other phytochromes of this group. Because BV has a vinyl side chain at ring A, the coupling mechanism must be different between Agp1 and plant phytochromes. It has been proposed that BV binds via its ring D to the protein (8Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (164) Google Scholar), but this assumption has not been confirmed by chemical studies. Chemically synthesized bilins have been used to characterize the chromophore pocket and to study photoconversion and physiological action of plant phytochromes (18Hanzawa H. Shinomura T. Inomata K. Kakiuchi T. Kinoshita H. Wada K. Furuya M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4725-4729Crossref PubMed Scopus (42) Google Scholar, 19Hanzawa H. Inomata K. Kinoshita H. Kakiuchi T. Jayasundera K.P. Sawamoto D. Ohta A. Uchida K. Wada K. Furuya M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3612-3617Crossref PubMed Scopus (34) Google Scholar, 20Lindner I. Knipp B. Braslavsky S.E. Gärtner W. Schaffner K. Angew. Chem. Int. Ed. Engl. 1998; 37: 1843-1846Crossref Scopus (33) Google Scholar). Here we used synthetic BV derivatives to test whether the ring A or ring D vinyl side chain is used for covalent BV attachment in Agp1. We also tested whether PCB binds covalently to the protein if a cysteine residue is introduced in the GAF domain of Agp1. Competition studies with BV and PCB were performed to further characterize the chromophore pocket of Agp1. Our findings have implications for the three-dimensional folding of the protein, the lyase mechanism, and the early evolution of phytochromes. Bilin Chromophores, Synthesis of Bilin Derivatives—BV was purchased from Frontier Scientific (Carnforth, UK). Phycocyanobilin was extracted from Spirulina geitlerie and purified via high pressure liquid chromatography (21Lamparter T. Esteban B. Hughes J. Eur. J. Biochem. 2001; 268: 4720-4730Crossref PubMed Scopus (99) Google Scholar). The synthesis of PΦB was performed as described (22Kakiuchi T. Kinoshita H. Inomata K. Synlett. 1999; SI: 901-904Crossref Scopus (36) Google Scholar). Synthesis of 3-Et-BV, 18-Et-BV, 3-18-Et-BV, and BV-4 followed protocols published for PΦB, PCB, and PCB derivatives (22Kakiuchi T. Kinoshita H. Inomata K. Synlett. 1999; SI: 901-904Crossref Scopus (36) Google Scholar, 23Kakiuchi T. Kato H. Jaysundera K.P. Higashi T. Watabe K. Sawamoto D. Kinoshita H. Inomata K. Chem. Lett. 1998; : Scopus Google Scholar, S. Jaysundera K.P. Kakiuchi T. Kinoshita H. Inomata K. Chem. Lett. 2001; : Scopus Google Scholar, D. Inomata K. Chem. Lett. 2001; : Scopus Google the be published Bilin were in The of were in and an of for BV D. The Academic Google Scholar) and for PCB Siegelman H.W. J. Chem. Scopus Google Scholar) at the in the region of the For 3-Et-BV, 18-Et-BV, 3-18-Et-BV, and we used the as for In the case of PΦB it was that the is to of Agp1 and the in which the for Agrobacterium phytochrome Agp1 has been into the was used (8Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (164) Google Scholar). The protein has an N-terminal The V249C mutant was with a site-directed mutagenesis to the of and are given in (8Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (164) Google Scholar). the protein were and the protein was with in and at and for were with a at The protein was to as by at For chromophore of were with to of bilin from the to a bilin of The Pr were this into Pfr was performed in the by with a photoconversion an as by at a was The were from both test for covalent of were with of to a of In general, was the chromophore was with Agp1 In the case of the V249C mutant, was 1 of chromophore the of a of the was of the were to a which been in 1 of was to the The protein with covalently bound bilin and is of A of this was also The protein at was used to both and from the in the it was whether chromophore with the protein. When covalent chromophore attachment was in the competition the are The proposed natural chromophore of Agp1 is BV (8Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (164) Google Scholar). BV, we tested the PCB and PΦB, with reduced ring A ring D side 3-Et-BV, 18-Et-BV, 3-18-Et-BV, and in which the ring D vinyl group of the is with the group of the for with the Agp1 protein. results in the of the Pr which is by into the protein, the of the of the chromophore to the in this region and that of the Fig. for BV and Fig. for the BV This was found for all tested 2, that all adducts with the Agp1 protein. In all adducts the photoconversion into the Pfr form with light. The of the Pr with the of bilin double that in the and BV-4 have double and adducts at and Both with double PCB and 3-18-Et-BV, adducts with the at and with double PΦB, 3-Et-BV, and 18-Et-BV, The of PΦB and were close and that of 18-Et-BV, that the ring D vinyl double bond more to the than the ring A vinyl double with Agp1 and Agp1 was with BV and BV The and chemical structure of the is given in The of BV was and the of the other bilins was the Agp1 V249C mutant was with PCB and the Pr chromophore and protein the photoconversion by and the Pr Pfr of of and in a into Pfr of the BV to a of the as from the The of with PCB with and PΦB with were close to BV, whereas all other at phytochrome the chromophore a to around its double bond (12Rüdiger W. Thümmler F. Kendrick R.E. Kronenberg G.H.M. Photomorphogenesis of Plants. 2nd Ed. Kluwer Academic Publishers, Dordrecht, The Netherlands1994: 51-69Google Scholar), but further for the are A that the chromophore is in the Pfr We have also the between the Pr and the Pfr of the The of was with and PCB of and the with PΦB and were whereas a high was with It that a ring D vinyl side chain the and that a ring A vinyl side chain that attachment of the was tested by followed by of protein and bilin the chromophore is covalently it with the protein from the whereas bound chromophore is from the protein by and the (8Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (164) Google Scholar). that are and the are shown in Fig. The were at the protein to for that during the the and of the at and The of the BV was during the in the region, an that be by during the or by with the The that most if not all BV was covalently attached to the protein. In the case of 18-Et-BV, both were of the chromophore were covalently bound the of the BV-4 adduct, it can be that of the chromophore were covalently attached covalently bound have a ring A vinyl side chain but by ring D side that a ring A ethyl or ethylidene side 3-Et-BV, 3-18-Et-BV, PCB, and PΦB, were bound to the protein. In all those the in the was the and Because of bilins from the the test (8Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (164) Google Scholar), of chromophore be to The noncovalent of in Agp1 is with CphB from the tested for Agp1 whether the of a cysteine in the GAF domain can also covalent attachment of PCB as was found for CphB (16Jorissen H.J. Quest B. Remberg A. Coursin T. Braslavsky S.E. Schaffner K. Tandeau de Marsac N. Gärtner W. Eur. J. Biochem. 2002; 269: 2662-2671Crossref PubMed Scopus (54) Google Scholar). The amino acid which is homologous to the chromophore-binding cysteine of plant phytochromes, was by and the purified protein was tested for PCB The a Pr of and was by as with the PCB of wild type the Pfr was also to and This that the ethylidene double bond of PCB, which is of the is as a of the of a covalent thioether attachment was confirmed by the whether two types of the chromophore-binding competition experiments were performed with BV and Agp1 was with PCB of and with additional BV In the the was with BV and with PCB of the chromophore the in the region this results from the and of bilin This finding that the pocket is used for both In both competition the Pr the of the chromophore and A of the of either chromophore and the Pr of either not the of the Pr not the that the bilin was by the bilin in both competition the the further the of both experiments a of not Because this is to the of the BV than to the PCB adduct, it that BV binds to Agp1 than PCB, but the results that covalent BV attachment not to an These findings the whether the covalent the biliverdin of Agp1. Other adducts are is by the PCB We covalent BV attachment with and additional The that were and were PCB not the of BV from the We also tested for chromophore competition When the PCB Agp1 was with to most of the phytochrome into the Pfr form and BV was to the a Pr at In this BV with PCB of the Pfr adduct, the of was by a reduction of the Pfr at the was the BV was to Pfr and PCB was to the The of the that was is by of Competition experiments with BV and PCB were also performed with the V249C mutant, which has one covalent site for either When the protein was with PCB, and BV was the of the Pr the of the BV Pr adduct, a was that during the for in Fig. These that PCB is by BV in the mutant protein. When V249C was with BV, and PCB was the of BV by PCB was PCB the Pr to lower and a at the of the PCB Pr a was at that at the of the Both competition experiments that PCB binds much to the mutant protein than BV and PCB binds to the mutant than to the wild type protein. Because in the mutant, either of both can form a covalent with the protein, two different can covalently bound to one protein as is from the of Fig. This assumption was confirmed by and the in which was with BV and with PCB chromophore than the that was with BV In plant phytochromes, the phytochromobilin chromophore is bound to a conserved cysteine within the GAF domain (12Rüdiger W. Thümmler F. Kendrick R.E. Kronenberg G.H.M. Photomorphogenesis of Plants. 2nd Ed. Kluwer Academic Publishers, Dordrecht, The Netherlands1994: 51-69Google Scholar). BV attachment has recently been shown for bacterial phytochromes (5Bhoo S.H. Davis S.J. Walker J. Karniol B. Vierstra R.D. Nature. 2001; 414: 776-779Crossref PubMed Scopus (253) Google Scholar, 8Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (164) Google Scholar), but it was which BV side group is used as attachment The this for Agrobacterium phytochrome Agp1 by different bilin all tested adducts that the photoconversion into the Pfr those bilins that have a ring A vinyl side chain were covalently attached to the protein. We that the ring A vinyl group forms a thioether bond with the cysteine 20 residue of Agp1. In general, the between the Pr and the Pfr was the ring A vinyl side chain was in the can that a covalently chromophore a for the Pfr form of Agp1. This assumption with an in which covalent BV attachment was by or by of the chromophore-binding cysteine (8Lamparter T. Michael N. Mittmann F. Esteban B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11628-11633Crossref PubMed Scopus (164) Google Scholar). the photocycle is possible with attached of Pfr to be covalent probably a coupling can to the chromophore into the the evolution of phytochromes, there was a from BV to that in the group of phytochromes have a conserved cysteine close to the N which at in Agp1 is used as a chromophore attachment the other species have a conserved chromophore-binding cysteine in the GAF domain. The between both not as shown by mutant in two different BV-binding phytochromes, CphB (16Jorissen H.J. Quest B. Remberg A. Coursin T. Braslavsky S.E. Schaffner K. Tandeau de Marsac N. Gärtner W. Eur. J. Biochem. 2002; 269: 2662-2671Crossref PubMed Scopus (54) Google Scholar) and Agrobacterium Agp1 the of a cysteine in the GAF domain was to covalent PCB attachment. phytochrome has one chromophore as shown by competition experiments for Agp1 Because BV binds with its ring A vinyl side chain to of and PCB binds with its ring A ethylidene side chain to of the protein must be in such a that amino acid 249 close to of Agp1. It is that both regions of the protein contribute to the of the chromophore pocket. a PAS domain is identified by structure in the N terminus of two bacterial phytochromes, Cph1 and (11Montgomery B.L. Lagarias J.C. Trends Plant Sci. 2002; 7: 357-366Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). The of Agp1 from to and the chromophore-binding of Agp1. In phytochrome A, the the region around amino acid which is for chromophore attachment S.H. T. Furuya M. J. Chem. 1997; Scopus Google Scholar). PAS often as contact I. M. Nature. PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), but in W. B. G. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar), photoactive protein W. B. G. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar), and M. K. Wada M. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar), this domain chromophore The domain in the N terminus of phytochromes could be of the chromophore pocket. It could be possible that in more phytochromes, this domain a chromophore pocket by its and that the GAF domain as which covalent chromophore attachment. The competition studies to an for why the chromophore site could have swapped from the N terminus into the GAF domain. covalently bound to BV was removed from the chromophore pocket by The competition by PCB was the BV was into the Pfr of bilin the photocycle of phytochromes. As as the PCB, as cyanobacteria the to this bilin as a phytochrome Phycocyanobilin has the that it can be bound to the protein, given that it is covalently bound to the GAF domain. in cyanobacteria pressure could phytochromes that have a cysteine in the GAF domain. We and for