Photocycle of the LOV-STAS protein from the pathogen Listeria monocytogenes.

Listeria monocytogenes, a food-borne bacterial pathogen causing significant human mortality, propagates by expressing genes in response to environmental signals, such as temperature and pH. Listeria gene (lmo0799) encodes a protein homologous to the Bacillus subtilis YtvA, which has a flavin-light, oxygen or voltage (LOV) domain and a Sulfate Transporters Anti-Sigma factor antagonist (STAS) output domain that regulates transcription-initiation factor Sigma B in the bacterial stress response upon exposure to light. This could be significant for the pathogenesis of listeriosis because Sigma B has been linked to virulence of Listeria, and the Listeria Lmo0799 protein has recently been identified as a virulence factor activated by blue light. We have cloned, expressed heterologously in Escherichia coli and purified the full-length LM-LOV-STAS protein. Although it exhibits photochemical activity similar to that of YtvA, LM-LOV-STAS lacks an almost universally conserved arginine in the flavin-binding site, as well as another positively charged residue, a lysine in YtvA. The absence of these positive charges was found to destabilize retention of the flavin mononucleotide (FMN) chromophore in the LM-LOV-STAS protein, particularly at higher temperatures. The unusual sequence of the LM-LOV-STAS protein alters both spectral features and activation/deactivation kinetics, potentially expanding the sensory capacity of this LOV domain, e.g. to detect light plus cold.

Structural water cluster as a possible proton acceptor in the adduct decay reaction of oat phototropin 1 LOV2 domain.

LOV domains (Light, Oxygen, Voltage) are the light-sensory modules of phototropins, the blue-light photoreceptor kinases in plants, and of a wide variety of flavoproteins found in all three domains of life. These 12 kDa modules bind a flavin chromophore (FMN or FAD) noncovalently and undergo a photochemical activation in which the sulfur atom of a conserved cysteine forms an adduct to the C(4a) carbon of the flavin. The adduct breaks spontaneously in a base-catalyzed reaction involving a rate-limiting proton-transfer step, regenerating the dark state in seconds. This photocycle involves chromophore and protein structural changes that activate the C-terminal serine/threonine kinase. Previous studies (Biochemistry 2007, 46, 7016-7021) showed that decreased hydration obtained at high glycerol concentrations stabilizes the adduct state in a manner similar to that attained at low temperatures, resulting in much longer adduct decay times. This kinetic effect was attributed to an increased protein rigidity that hindered structural fluctuations necessary for the decay reaction. In this work, we studied the adduct decay kinetics of oat phototropin 1 (phot1) LOV2 at varying hydration using a specially designed chamber that allowed for measurement of UV-visible and FTIR spectra of the same samples. Therefore, we obtained LOV protein concentrations, adduct decay kinetics, and the different populations of bound water by deconvolution of the broad water absorption peak around 3500 cm(-1). A linear dependence of the adduct decay rate constant on the concentration of double and triple hydrogen-bonded waters strongly suggests that the adduct decay is a pseudo-first-order reaction in which both the adduct and the strongly bound waters are reactants. We suggest that a cluster of strongly bound water functions as the proton acceptor in the rate-limiting step of adduct decay.