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J. Biol. Chem., Vol. 278, Issue 13, 10973-10982, March 28, 2003

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Blue Light Perception in Plants
DETECTION AND CHARACTERIZATION OF A LIGHT-INDUCED NEUTRAL FLAVIN RADICAL IN A C450A MUTANT OF PHOTOTROPIN^*

Christopher W. M. Kay, Erik Schleicher^§, Andreas Kuppig, Heidi Hofner^§, Wolfhart Rüdiger^¶, Michael Schleicher, Markus Fischer^§, Adelbert Bacher^§, Stefan Weber^**, and Gerald Richter^§

From the  Institut für Experimentalphysik, Freie Universität Berlin, Arnimallee 14, 14195 Berlin, Germany, ^§ Lehrstuhl Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching, Germany, ^¶ Institut für Botanik, Ludwig-Maximilians-Universität München, Menzinger Strasse 64, 80638 München, Germany, and  Institut für Zellbiologie, Ludwig-Maximilians-Universität München, Schillerstrasse 42, 80336 München, Germany

The LOV2 domain of Avena sativa phototropin and its C450A mutant were expressed as recombinant fusion proteins and were examined by optical spectroscopy, electron paramagnetic resonance, and electron-nuclear double resonance. Upon irradiation (420-480 nm), the LOV2 C450A mutant protein gave an optical absorption spectrum characteristic of a flavin radical even in the absence of exogenous electron donors, thus demonstrating that the flavin mononucleotide (FMN) cofactor in its photogenerated triplet state is a potent oxidant for redox-active amino acid residues within the LOV2 domain. The FMN radical in the LOV2 C450A mutant is N(5)-protonated, suggesting that the local pH close to the FMN is acidic enough so that the cysteine residue in the wild-type protein is likely to be also protonated. An electron paramagnetic resonance analysis of the photogenerated FMN radical gave information on the geometrical and electronic structure and the environment of the FMN cofactor. The experimentally determined hyperfine couplings of the FMN radical point to a highly restricted delocalization of the unpaired electron spin in the isoalloxazine moiety. In the light of these results a possible radical-pair mechanism for the formation of the FMN-C(4a)-cysteinyl adduct in LOV domains is discussed.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF544403.

This paper is dedicated to the memory of Vincent Massey.

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