Light-induced Electron Transfer in Arabidopsis Cryptochrome-1 Correlates with in Vivo Function*

Cryptochromes are blue light-activated photoreceptors found in multiple organisms with significant similarity to photolyases, a class of light-dependent DNA repair enzymes. Unlike photolyases, cryptochromes do not repair DNA and instead mediate blue light-dependent developmental, growth, and/or circadian responses by an as yet unknown mechanism of action. It has recently been shown that Arabidopsis cryptochrome-1 retains photolyase-like photoreduction of its flavin cofactor FAD by intraprotein electron transfer from tryptophan and tyrosine residues. Here we demonstrate that substitution of two conserved tryptophans that are constituents of the flavin-reducing electron transfer chain in Escherichia coli photolyase impairs light-induced electron transfer in the Arabidopsis cryptochrome-1 photoreceptor in vitro. Furthermore, we show that these substitutions result in marked reduction of light-activated autophosphorylation of cryptochrome-1 in vitro and of its photoreceptor function in vivo, consistent with biological relevance of the electron transfer reaction. These data support the possibility that light-induced flavin reduction via the tryptophan chain is the primary step in the signaling pathway of plant cryptochrome.

[1]  C. Green Cryptochromes: Tail-ored for Distinct Functions , 2004, Current Biology.

[2]  Chad A Brautigam,et al.  Structure of the photolyase-like domain of cryptochrome 1 from Arabidopsis thaliana. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[3]  M. Byrdin,et al.  Intraprotein electron transfer and proton dynamics during photoactivation of DNA photolyase from E. coli: review and new insights from an "inverse" deuterium isotope effect. , 2004, Biochimica et biophysica acta.

[4]  Chentao Lin,et al.  Cryptochrome structure and signal transduction. , 2003, Annual review of plant biology.

[5]  T. Mockler,et al.  Blue Light–Dependent in Vivo and in Vitro Phosphorylation of Arabidopsis Cryptochrome 1 Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.013011. , 2003, The Plant Cell Online.

[6]  Markus Mueller,et al.  Novel ATP-binding and autophosphorylation activity associated with Arabidopsis and human cryptochrome-1. , 2003, European journal of biochemistry.

[7]  M. Byrdin,et al.  Dissection of the triple tryptophan electron transfer chain in Escherichia coli DNA photolyase: Trp382 is the primary donor in photoactivation , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[8]  Baldissera Giovani,et al.  Light-induced electron transfer in a cryptochrome blue-light photoreceptor , 2003, Nature Structural Biology.

[9]  Aziz Sancar,et al.  Structure and function of DNA photolyase and cryptochrome blue-light photoreceptors. , 2003, Chemical reviews.

[10]  Minoru Kanehisa,et al.  Identification of a new cryptochrome class. Structure, function, and evolution. , 2003, Molecular cell.

[11]  N. Mataga,et al.  Femtosecond fluorescence dynamics of flavoproteins: Comparative studies on flavodoxin, its site-directed mutants, and riboflavin binding protein regarding ultrafast electron transfer in protein nanospaces , 2002 .

[12]  Paul Galland,et al.  Action Spectrum for Cryptochrome-Dependent Hypocotyl Growth Inhibition in Arabidopsis1 , 2002, Plant Physiology.

[13]  T. Todo,et al.  Photoactivation of the flavin cofactor in Xenopus laevis (6–4) photolyase: Observation of a transient tyrosyl radical by time-resolved electron paramagnetic resonance , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[14]  O. Froy,et al.  Redox potential: differential roles in dCRY and mCRY1 functions. , 2002, Current biology : CB.

[15]  Haisun Zhu,et al.  A putative flavin electron transport pathway is differentially utilized in Xenopus CRY1 and CRY2 , 2001, Current Biology.

[16]  A. Zewail,et al.  Femtosecond dynamics of flavoproteins: Charge separation and recombination in riboflavine (vitamin B2)-binding protein and in glucose oxidase enzyme , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[17]  T. Carell,et al.  The mechanism of action of DNA photolyases. , 2001, Current opinion in chemical biology.

[18]  J. Christie,et al.  Blue Light Sensing in Higher Plants* , 2001, The Journal of Biological Chemistry.

[19]  Yan Liu,et al.  The C Termini of Arabidopsis Cryptochromes Mediate a Constitutive Light Response , 2000, Cell.

[20]  A. Eker,et al.  Intraprotein radical transfer during photoactivation of DNA photolyase , 2000, Nature.

[21]  Christopher C. Moser,et al.  Natural engineering principles of electron tunnelling in biological oxidation–reduction , 1999, Nature.

[22]  A. Stuchebrukhov,et al.  Pathways of electron transfer in Escherichia coli DNA photolyase: Trp306 to FADH. , 1999, Biophysical journal.

[23]  A. Cashmore,et al.  Chimeric Proteins between cry1 and cry2 Arabidopsis Blue Light Photoreceptors Indicate Overlapping Functions and Varying Protein Stability , 1998, Plant Cell.

[24]  M. Ahmad,et al.  Mutations throughout an Arabidopsis blue-light photoreceptor impair blue-light-responsive anthocyanin accumulation and inhibition of hypocotyl elongation. , 1995, The Plant journal : for cell and molecular biology.

[25]  M. Ahmad,et al.  Association of flavin adenine dinucleotide with the Arabidopsis blue light receptor CRY1 , 1995, Science.

[26]  J. Deisenhofer,et al.  Crystal structure of DNA photolyase from Escherichia coli. , 1995, Science.

[27]  A. Sancar,et al.  Active site of DNA photolyase: tryptophan-306 is the intrinsic hydrogen atom donor essential for flavin radical photoreduction and DNA repair in vitro. , 1991, Biochemistry.

[28]  P. Heelis The photophysical and photochemical properties of flavins (isoalloxazines) , 1982 .

[29]  V. Massey,et al.  On the existence of spectrally distinct classes of flavoprotein semiquinones. A new method for the quantitative production of flavoprotein semiquinones. , 1966, Biochemistry.