Intramolecular proton transfers and structural changes during the photocycle of the LOV2 domain of phototropin 1.
The phototropins are a family of membrane-associated flavoproteins that function as the primary blue light receptors regulating phototropism, chloroplast movements, stomatal opening, and leaf expansion in plants. Phot1, a member of this family, contains two FMN-binding domains, LOV1 and LOV2, within the N-terminal region and a C-terminal serine-threonine protein kinase domain. Light irradiation of oat phot1 LOV2 produces a cysteinyl adduct (Cys-39) at the flavin C(4a) position, which decays thermally back to the dark state. We measured pH and isotope effects on the photocycle. Between pH 3.7 and 9.5, adduct formation showed minimal pH dependence, and adduct decay showed only slight pH dependence, indicating that the pK values of mechanistically relevant groups are outside this range. LOV2 showed a nearly 5-fold slowing of adduct formation in D(2)O relative to H(2)O, indicating that the rate-limiting step involves proton transfer(s). Light-induced changes in the far UV CD spectrum of LOV2 revealed putative protein structural perturbations. The light minus dark CD difference spectrum resembles an inverted alpha-helix spectrum, suggesting that alpha-helicity is reversibly lost upon light irradiation. Decay kinetics for CD spectral changes in the far UV region occur at the same rate as those in the visible region, indicating synchronous relaxation of protein and chromophore structures.
Corchnoy
Stephanie B
SB
Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064, USA.
Swartz
Trevor E
TE
Lewis
James W
JW
Szundi
Istvan
I
Briggs
Winslow R
WR
Bogomolni
Roberto A
RA
D000595:Amino Acid Sequence; D018554:Avena; D001665:Binding Sites; D002942:Circular Dichroism; D056931:Cryptochromes; D003545:Cysteine / Q000737:chemistry; D029721:Drosophila Proteins; D005136:Eye Proteins; D005486:Flavin Mononucleotide / Q000737:chemistry*; D005420:Flavoproteins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008969:Molecular Sequence Data; D017956:Photoreceptor Cells, Invertebrate; D010940:Plant Proteins / Q000737:chemistry*; D011522:Protons; D043562:Receptors, G-Protein-Coupled
D056931:Cryptochromes; D029721:Drosophila Proteins; D005136:Eye Proteins; D005420:Flavoproteins; D010940:Plant Proteins; D011522:Protons; D043562:Receptors, G-Protein-Coupled; C119828:cry protein, Drosophila; D005486:Flavin Mononucleotide; D003545:Cysteine