An extended hydrogen-bonding (HB) network stabilizes the isoalloxazine ring of the flavin mononucleotide (FMN) chromophore within the photosensing LOV domain of blue-light protein receptors, via interactions between the C(2)═O, N(3)H, C(4)═O, and N(5) groups and conserved glutamine and asparagine residues. In this work we studied the influence of the HB network on the efficiency, kinetics, and energetics of a LOV protein photocycle, involving the reversible formation of a FMN-cysteine covalent adduct. The following results were found for mutations of the conserved amino acids N94, N104, and Q123 in the Bacillus subtilis LOV protein YtvA: (i) Increased (N104D, N94D) or strongly reduced (N94A) rate of adduct formation; this latter mutation extends the lifetime of the flavin triplet state, i.e., adduct formation, more than 60-fold, from 2 μs for the wild-type (WT) protein to 129 μs. (ii) Acceleration of the overall photocycle for N94S, N94A, and Q123N, with recovery lifetimes 20, 45, and 85 times faster than for YtvA-WT, respectively. (iii) Slight modifications of FMN spectral features, correlated with the polarization of low-energy transitions. (iv) Strongly reduced (N94S) or suppressed (Q123N) structural volume changes accompanying adduct formation, as determined by optoacoustic spectroscopy. (v) Minor effects on the quantum yield, with the exception of a considerable reduction for Q123N, i.e., 0.22 vs 0.49 for YtvA-WT. The data stress the importance of the HB network in modulating the photocycle of LOV domains, while at the same time establishing a link with functional responses.

The gene slr1393 from Synechocystis sp. PCC6803 encodes a protein composed of three GAF domains, a PAS domain, and a histidine kinase domain. GAF3 is the sole domain able to bind phycocyanobilin (PCB) as chromophore and to accomplish photochemistry: switching between a red-absorbing parental and a green-absorbing photoproduct state (λmax =649 and 536 nm, respectively). Conversions in both directions were followed by time-resolved absorption spectroscopy with the separately expressed GAF3 domain of Slr1393. Global fit analysis of the recorded absorbance changes yielded three lifetimes (3.2 μs, 390 μs, and 1.5 ms) for the red-to-green conversion, and 1.2 μs, 340 μs, and 1 ms for the green-to-red conversion. In addition to the wild-type (WT) protein, 24 mutated proteins were studied spectroscopically. The design of these site-directed mutations was based on sequence alignments with related proteins and by employing the crystal structure of AnPixJg2 (PDB ID: 3W2Z), a Slr1393 orthologous from Anabaena sp. PCC7120. The structure of AnPixJg2 was also used as template for model building, thus confirming the strong structural similarity between the proteins, and for identifying amino acids to target for mutagenesis. Only amino acids in close proximity to the chromophore were exchanged, as these were considered likely to have an impact on the spectral and dynamic properties. Three groups of mutants were found: some showed absorption features similar to the WT protein, a second group showed modified absorbance properties, and the third group had lost the ability to bind the chromophore. The most unexpected result was obtained for the exchange at residue 532 (N532Y). In vivo assembly yielded a red-absorbing, WT-like protein. Irradiation, however, not only converted it into the green-absorbing form, but also produced a 660 nm, further-red-shifted absorbance band. This photoproduct was fully reversible to the parental form upon green light irradiation.

Genome screening of the cyanobacterium Microcoleus chthonoplastes PCC 7420 identified a gene encoding a protein (483 amino acids, 54.2 kDa in size) characteristic of a BL (blue light)-regulated adenylate (adenylyl) cyclase function. The photoreceptive part showed signatures of a LOV (light, oxygen, voltage) domain. The gene product, mPAC (Microcoleus photoactivated adenylate cyclase), exhibited the LOV-specific three-peaked absorption band (λmax=450 nm) and underwent conversion into the photoadduct form (λmax=390 nm) upon BL-irradiation. The lifetime for thermal recovery into the parent state was determined as 16 s at 20°C (25 s at 11°C). The adenylate cyclase function showed a constitutive activity (in the dark) that was in-vitro-amplified by a factor of 30 under BL-irradiation. Turnover of the purified protein at saturating light and pH 8 is estimated to 1 cAMP/mPAC per s at 25°C (2 cAMP/mPAC per s at 35°C). The lifetime of light-activated cAMP production after a BL flash was ~14 s at 20°C. The temperature optimum was determined to 35°C and the pH optimum to 8.0. The value for half-maximal activating light intensity is 6 W/m2 (at 35°C). A comparison of mPAC and the BLUF (BL using FAD) protein bPAC (Beggiatoa PAC), as purified proteins and expressed in Xenopus laevis oocytes, yielded higher constitutive activity for mPAC in the dark, but also when illuminated with BL.

Flavin-binding light, oxygen, and voltage (LOV) domains are UVA/blue-light-sensing protein units that form a reversible flavin mononucleotide-cysteine adduct upon light induction. In their dark-adapted state, LOV domains exhibit the typical spectral features of fully oxidized riboflavin derivatives. A survey on the absorption spectra of various LOV domains revealed that the UVA spectral range is the most variable region (whereas the absorption band at 450 nm is virtually unchanged), showing essentially two distinct patterns found in plant phototropin LOV1 and LOV2 domains, respectively. In this work, we have identified a residue directly interacting with the isoalloxazine methyl group at C(7a) as the major UVA spectral tuner. In YtvA from Bacillus subtilis, this amino acid is threonine 30, and its mutation into apolar residues converts the LOV2-like spectrum of native YtvA into a LOV1-like pattern. Mutation T30A also accelerates the photocycle ca. 4-fold. Together with control mutations at different positions, our results experimentally confirm the previously calculated direction of the transition dipole moment for the UVA ππ* state and identify the mechanisms underlying spectral tuning in the LOV domains.

We introduce a novel fluorescent reporter with potential for super-resolution microscopy, based on the bacterial photoreceptor YtvA. YtvA (from Bacillus subtilis) comprises a photosensitive flavin-based LOV domain, efficiently photo-switchable between fluorescent and non-fluorescent states. We demonstrate Fluorescence PhotoActivation Localization Microscopy (FPALM) studies of live Escherichia coli cells, expressing YtvA molecules.