'American Society for Biochemistry & Molecular Biology (ASBMB)'
Doi
Abstract
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 photoinduced 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.Fil: von Stetten, David. Technische Universität Berlin; AlemaniaFil: Seibeck, Sven. Freie Universität Berlin.; AlemaniaFil: Michael, Norbert. Freie Universität Berlin.; AlemaniaFil: Scheerer, Patrick. Charité Universitätsmedizin Berlin; AlemaniaFil: Mroginski, Maria Andrea. Technische Universität Berlin; AlemaniaFil: Murgida, Daniel Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Technische Universität Berlin; AlemaniaFil: Krauss, Norbert. Freie Universität Berlin.; AlemaniaFil: Heyn, Maarten P.. Charité Universitätsmedizin Berlin; AlemaniaFil: Hildebrandt, Peter. Technische Universität Berlin; AlemaniaFil: Borucki, Berthold. Freie Universität Berlin.; AlemaniaFil: Lamparter, Tilman. Freie Universität Berlin.; Alemani