A Red/Green Cyanobacteriochrome Sustains Its Color Despite a Change in the Bilin Chromophore's Protonation State.
The second GAF domain of AnPixJ, AnPixJg2, a bilin-binding protein from the cyanobacterium Anabaena PCC 7120, undergoes a photoinduced interconversion between a red-absorbing state, Pr, and a green-absorbing state, Pg. Combining ultraviolet-vis (UV-vis), infrared, resonance Raman (RR), and magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, we have studied this cyanobacteriochrome (CBCR) assembled with phycocyanobilin (PCB) either in vivo or in vitro. In both assembly routes, the spectroscopic data of the Pr state reveal nearly identical chromophore structures with a protonated (cationic) bilin. However, unlike the native (in vivo assembly) Pg photoproduct, in which the bilin retains protonation, the Pg generated from the in vitro-assembled AnPixJg2 harbors a deprotonated (neutral) bilin chromophore at pH 7.8. IR difference spectroscopy further reveals the transfer of a proton from the bilin to a side-chain carboxylate on an amino acid, probably Asp291. Besides the change in protonation state, the bilin structure is very similar in the in vitro- and in vivo-assembled Pg photoproducts. The chromophore of the in vitro Pg becomes protonated when the pH is increased to 10, presumably because of a partial reversal of protein misfolding. Most remarkably, the electronic transitions remain unchanged and are very similar to those of the native Pg. Thus, bilin protonation is not a key parameter for controlling the energies of the electronic transitions in AnPixJg2. Possible alternative molecular mechanisms for color tuning are discussed.
Leids Instituut voor Chemisch Onderzoek, Universiteit Leiden , 2300 RA Leiden, The Netherlands.|Institut für Analytische Chemie, Universität Leipzig , Linnéstraße 3, D-04103 Leipzig, Germany.
Chen
C
Song
Technische Universität Berlin , Institut für Chemie, Sekr. PC14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
Francisco
F
Velazquez Escobar
Max-Planck-Institut für Chemische Energiekonversion , Stiftstraße 34-36, D-45470 Mülheim an der Ruhr, Germany.
Xiu-Ling
XL
Xu
Department of Life Sciences (Biology), Graduate School of Art and Sciences, University of Tokyo , Komaba 3-8-1, Meguro, Tokyo 153-8902, Japan.|Department of Biological Science, Faculty of Science, Shizuoka University , Shizuoka 422-8529, Japan.|Precursory Research for Embryonic Science and Technology (PRESTO) , Meguro, Tokyo 153-8902, Japan.
Rei
R
Narikawa
Department of Life Sciences (Biology), Graduate School of Art and Sciences, University of Tokyo , Komaba 3-8-1, Meguro, Tokyo 153-8902, Japan.|Japan Science and Technology Agency (JST), Core Research for Evolutionary Science and Technology (CREST) , Meguro, Tokyo 153-8902, Japan.
Masahiko
M
Ikeuchi
Technische Universität Berlin , Institut für Chemie, Sekr. PC14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
Friedrich
F
Siebert
Max-Planck-Institut für Chemische Energiekonversion , Stiftstraße 34-36, D-45470 Mülheim an der Ruhr, Germany.
Wolfgang
W
Gärtner
Institut für Analytische Chemie, Universität Leipzig , Linnéstraße 3, D-04103 Leipzig, Germany.
Jörg
J
Matysik
Technische Universität Berlin , Institut für Chemie, Sekr. PC14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
Peter
P
Hildebrandt
D017033:Anabaena / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001654:Bile Pigments / Q000737:chemistry / Q000378:metabolism*; D003116:Color; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D052980:Phycobilins / Q000737:chemistry / Q000378:metabolism*; D010798:Phycocyanin / Q000737:chemistry / Q000378:metabolism*; D010834:Phytochrome; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011522:Protons; D013055:Spectrophotometry, Infrared; D013056:Spectrophotometry, Ultraviolet
D001426:Bacterial Proteins; D001654:Bile Pigments; D052980:Phycobilins; D011522:Protons; D010798:Phycocyanin; D010834:Phytochrome; C011069:phycocyanobilin