Intramolecular Proton Transfers and Structural Changes during the Photocycle of the LOV2 Domain of Phototropin 1

Stephanie B Corchnoy,Trevor E Swartz,James W Lewis,Istvan Szundi,Winslow R Briggs,Roberto A Bogomolni

doi:10.1074/jbc.m209119200

Stephanie B Corchnoy, Trevor E Swartz + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m209119200

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Abstract

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.

Highlights

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
The phototropins are a family of blue light receptors that are responsible for phototropism (1, 2) and are involved in lightinduced chloroplast movements (3) as well as blue light-stimulated stomatal opening (4) and leaf expansion in higher plants (5)
Blue light irradiation excites the FMN chromophore to a triplet state that absorbs maximally around 660 nm (15), resulting in electronic redistribution that increases the basicity of N-5 of the FMN chromophore (16)

Summary

Introduction

The phototropins are a family of blue light receptors that are responsible for phototropism (1, 2) and are involved in lightinduced chloroplast movements (3) as well as blue light-stimulated stomatal opening (4) and leaf expansion in higher plants (5). The phototropin phot (9, 10), which becomes autophosphorylated in response to blue light, is a membrane-associated protein that contains two 12-kDa, FMN-binding LOV (light, oxygen, voltage) (11, 12) domains (LOV1 and LOV2) in its N-terminal region and a typical serine-threonine kinase do-. Adduct decay for the LOV2 domain of oat phot has been shown to be 3 times slower in D2O than in H2O (15), indicating that proton transfer reactions, probably involving at least N-5, are rate-limiting components of the back reaction. Negligible protein motions were detected during the photocycle in crystals of the LOV2 domain of fern phy (19), whereas major shifts in the amide proton region were reported in a one-dimensional NMR study of the LOV2 photocycle of oat phot (18)

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