Functional Characterization of a LOV‐Histidine Kinase Photoreceptor from Xanthomonas citri subsp. citri

Abstract

The blue-light (BL) absorbing protein Xcc-LOV from Xanthomonas citri subsp. citri is composed of a LOV-domain, a histidine kinase (HK) and a response regulator. Spectroscopic characterization of Xcc-LOV identified intermediates and kinetics of the protein's photocycle. Measurements of steady state and time-resolved fluorescence allowed determination of quantum yields for triplet (ΦT = 0.68 ± 0.03) and photoproduct formation (Φ390 = 0.46 ± 0.05). The lifetime for triplet decay was determined as τT = 2.4-2.8 μs. Fluorescence of tryptophan and tyrosine residues was unchanged upon light-to-dark conversion, emphasizing the absence of significant conformational changes. Photochemistry was blocked upon cysteine C76 (C76S) mutation, causing a seven-fold longer lifetime of the triplet state (τT = 16-18.5 μs). Optoacoustic spectroscopy yielded the energy content of the triplet state. Interestingly, Xcc-LOV did not undergo the volume contraction reported for other LOV domains within the observation time window, although the back-conversion into the dark state was accompanied by a volume expansion. A radioactivity-based enzyme function assay revealed a larger HK activity in the lit than in the dark state. The C76S mutant showed a still lower enzyme function, indicating the dark state activity being corrupted by a remaining portion of the long-lived lit state.

Quantum Mechanics/Molecular Mechanics Study on the Photoreactions of Dark‐ and Light‐Adapted States of a Blue‐Light YtvA LOV Photoreceptor

“…Theu se of 400 nm excitation (71.5 kcal mol À1 )i n the experimental study [25] implies that only the T 1 transition states are readily accessible energetically,a nd hence the hydrogen transfer must occur on the lowest triplet surface. Consequently,the excited-state hydrogen transfer will involve an S 1 !T 1 intersystem crossing, consistent with previous experimental [10][11][12]25] and theoretical viewpoints. [28,29,33] The computed S 1 /T 1 spin-orbit coupling is rather small at the reactant geometries,a pproximately 0.1 cm À1 at the QM-…”

“…Due to small spin-orbit couplings in the reactant region, the overall photochemical reaction is rather slow in the dark-adapted state (microsecond timescale). [5,8,11,25] Thep hotochemistry of the light-adapted state is different and does not involve the T 1 state.The S 1 C4a-Sbond fission and the subsequent S 0 recombination are both essentially barrierless and thus ultrafast (ca. 0.3 and 1.1 ps, respectively [25] ).…”

“…Thel ight-adapted signaling state of light-activated LOVp hotoreceptors is generated through the formation of af lavin-(C4a)-cysteinyl covalent bond with ac onserved cysteine residue in the active site of LOVd omains (Figure 1). [5,6] In the past decade,t he excited-state dynamics of the dark-adapted state of LOVp hotoreceptors has been thoroughly investigated experimentally [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] while there have been less such studies on the light-adapted state.…”

Bacterial Photosensory Proteins and Their Role in Plant–pathogen Interactions

“…In fact, Xcc‐LOV is a legitimate photoreceptor capable of absorbing blue light and initiates a canonical LOV photocycle . In this protein, photochemistry does not take place in C76 mutations; confirming the involvement of this residue in the generation of the covalent photoadduct essential for photochemistry . The mechanism by which the Xcc‐LOV photoreceptor is activated upon an incoming blue light photon to start a signal transduction cascade has been elucidated by a complete spectroscopic analysis.…”

“…The LOV protein of Xcc (Xcc‐LOV) presents an N‐terminal LOV domain associated with a C‐terminal histidine kinase (HK) domain and a response regulator (RR) domain (hybrid HK‐RR). The amino acid sequence of Xcc‐LOV presents all the essential amino acids for LOV photochemistry, including the characteristic GXNCRFLQ motif containing the conserved cysteine residue involved in the covalent adduct formation upon blue light absorption (C76 in Xcc) . In fact, Xcc‐LOV is a legitimate photoreceptor capable of absorbing blue light and initiates a canonical LOV photocycle .…”