Animal type 1 cryptochromes. Analysis of the redox state of the flavin cofactor by site-directed mutagenesis.
It has recently been realized that animal cryptochromes (CRYs) fall into two broad groups. Type 1 CRYs, the prototype of which is the Drosophila CRY, that is known to be a circadian photoreceptor. Type 2 CRYs, the prototypes of which are human CRY 1 and CRY 2, are known to function as core clock proteins. The mechanism of photosignaling by the Type 1 CRYs is not well understood. We recently reported that the flavin cofactor of the Type 1 CRY of the monarch butterfly may be in the form of flavin anion radical, FAD(*-), in vivo. Here we describe the purification and characterization of wild-type and mutant forms of Type 1 CRYs from fruit fly, butterfly, mosquito, and silk moth. Cryptochromes from all four sources contain FAD(ox) when purified, and the flavin is readily reduced to FAD(*-) by light. Interestingly, mutations that block photoreduction in vitro do not affect the photoreceptor activities of these CRYs, but mutations that reduce the stability of FAD(*-) in vitro abolish the photoreceptor function of Type 1 CRYs in vivo. Collectively, our data provide strong evidence for functional similarities of Type 1 CRYs across insect species and further support the proposal that FAD(*-) represents the ground state and not the excited state of the flavin cofactor in Type 1 CRYs.
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599.
Nuri
N
Öztürk
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599.
Sang-Hun
SH
Song
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599.
Christopher P
CP
Selby
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599. Electronic address: Aziz_Sancar@med.unc.edu.
Aziz
A
Sancar
D000818:Animals; D003001:Cloning, Molecular; D056931:Cryptochromes; D004330:Drosophila; D004926:Escherichia coli / Q000378:metabolism; D005415:Flavins / Q000737:chemistry*; D005420:Flavoproteins / Q000737:chemistry*; D006801:Humans; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010777:Photochemistry / Q000379:methods; D013053:Spectrophotometry; D014364:Tryptophan / Q000737:chemistry
D056931:Cryptochromes; D005415:Flavins; D005420:Flavoproteins; D014364:Tryptophan; D010100:Oxygen