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 pK(a) 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.
von Stetten
David
D
Institut für Chemie, Technische Universität Berlin, Sekretariat PC14, Strasse des 17 Juni 135, D-10623 Berlin, Germany.
Seibeck
Sven
S
Michael
Norbert
N
Scheerer
Patrick
P
Mroginski
Maria Andrea
MA
Murgida
Daniel H
DH
Krauss
Norbert
N
Heyn
Maarten P
MP
Hildebrandt
Peter
P
Borucki
Berthold
B
Lamparter
Tilman
T
D016960:Agrobacterium tumefaciens / Q000378:metabolism*; D001224:Aspartic Acid / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D002845:Chromatography; D003001:Cloning, Molecular; D017124:Conserved Sequence; D006639:Histidine / Q000737:chemistry*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008968:Molecular Conformation; D009154:Mutation; D010834:Phytochrome / Q000737:chemistry*; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D010834:Phytochrome; D001224:Aspartic Acid; D006639:Histidine