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J. Biol. Chem., Vol. 283, Issue 18, 12212-12226, May 2, 2008

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Mutational Analysis of Deinococcus radiodurans Bacteriophytochrome Reveals Key Amino Acids Necessary for the Photochromicity and Proton Exchange Cycle of Phytochromes^*

Jeremiah R. Wagner¹², Junrui Zhang¹, David von Stetten¹, Mina Günther, Daniel H. Murgida, Maria Andrea Mroginski, Joseph M. Walker, Katrina T. Forest^¶, Peter Hildebrandt³, and Richard D. Vierstra⁴

From the Departments of Genetics and ^¶Bacteriology, University of Wisconsin, Madison, Wisconsin 53706 and Technische Universität Berlin, D-10623 Berlin, Germany

The ability of phytochromes (Phy) to act as photointerconvertible light switches in plants and microorganisms depends on key interactions between the bilin chromophore and the apoprotein that promote bilin attachment and photointerconversion between the spectrally distinct red light-absorbing Pr conformer and far red light-absorbing Pfr conformer. Using structurally guided site-directed mutagenesis combined with several spectroscopic methods, we examined the roles of conserved amino acids within the bilin-binding domain of Deinococcus radiodurans bacteriophytochrome with respect to chromophore ligation and Pr/Pfr photoconversion. Incorporation of biliverdin IX (BV), its structure in the Pr state, and its ability to photoisomerize to the first photocycle intermediate are insensitive to most single mutations, implying that these properties are robust with respect to small structural/electrostatic alterations in the binding pocket. In contrast, photoconversion to Pfr is highly sensitive to the chromophore environment. Many of the variants form spectrally bleached Meta-type intermediates in red light that do not relax to Pfr. Particularly important are Asp-207 and His-260, which are invariant within the Phy superfamily and participate in a unique hydrogen bond matrix involving the A, B, and C pyrrole ring nitrogens of BV and their associated pyrrole water. Resonance Raman spectroscopy demonstrates that substitutions of these residues disrupt the Pr to Pfr protonation cycle of BV with the chromophore locked in a deprotonated Meta-R_c-like photoconversion intermediate after red light irradiation. Collectively, the data show that a number of contacts contribute to the unique photochromicity of Phy-type photoreceptors. These include residues that fix the bilin in the pocket, coordinate the pyrrole water, and possibly promote the proton exchange cycle during photoconversion.

Received for publication, November 14, 2007 , and in revised form, January 10, 2008.

^* This work was supported by National Science Foundation Grants MCB 04240 62 and 0719153 (to R. D. V. and K. T. F.) and Deutsche Forschungsgemeinschaft Grant SFB498 (to P. H. and M. A. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.

¹ These authors contributed equally to this work.

² Present address: Dept. of Biology, Beloit College, Beloit, WI 53511.

³ To whom correspondence may be addressed: Technische Universität Berlin, Institut für Chemie, Max-Volmer-Laboratorium für Biophysikalische Chemie, Sekr. PC 14, Strasse des 17, Juni 135, D-10623 Berlin, Germany. Tel.: 49-30-314-21419; Fax: 49-30-314-21122; E-mail: hildebrandt{at}chem.tu-berlin.de. ⁴ To whom correspondence may be addressed: Dept. of Genetics, 425-G Henry Mall, University of Wisconsin, Madison, WI 53706. Tel.: 608-262-8215; Fax: 608-262-2976; E-mail: vierstra{at}wisc.edu.

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