Primary Photochemistry of the Dark- and Light-Adapted States of the YtvA Protein from Bacillus subtilis

Abstract

The primary (100 fs to 10 ns) and secondary (10 ns to 100 μs) photodynamics in the type II light-oxygen-voltage (LOV) domain from the blue light YtvA photoreceptor extracted from Bacillus subtilis were explored with transient absorption spectroscopy. The photodynamics of full-length YtvA were characterized after femtosecond 400 nm excitation of both the dark-adapted D447 state and the light-adapted S390 state. The S390 state relaxes on a 43 min time scale at room temperature back into D447, which is weakly accelerated by the introduction of imidazole. This is ascribed to an obstructed cavity in YtvA that hinders access to the embedded FMN chromophore and is more open in type I LOV domains. The primary photochemistry of dark-adapted YtvA is qualitatively similar to that of the type I LOV domains, including AsLOV2 from Avena sativa, but exhibits an appreciably higher (60% greater) terminal triplet yield, estimated near the maximal ΦISC value of ≈78%; the other 22% decays via non-triplet-generating fluorescence. The subsequent secondary dynamics are inhomogeneous, with three triplet populations co-evolving: the faster-decaying (I)T* population (38% occupancy) with a 200 ns decay time is nonproductive in generating the S390 adduct state, a slower (II)T* population (57% occupancy) exhibits a high yield (Φadduct ≈ 100%) in generating S390 and a third (5%) (III)T*population persists (>100 μs) with unresolved photoactivity. The ultrafast photoswitching dynamics of the S390 state appreciably differ from those previously resolved for the type I AcLOV2 domain from Adiantum capillus-veneris [Kennis, J. T., et al. (2004) J. Am. Chem. Soc. 126, 4512], with a low-yield dissociation (Φdis ≈ 2.5%) reaction, which is due to an ultrafast recombination reaction, following photodissociation, and is absent in AcLOV2, which results in the increased photoswitching activity of the latter domain.

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

“…This diradical nature conforms with earlier experimental notions [6,10] and theoretical calculations for phototropin-LOV1 [28,29] but not with the recent experimental suggestion of ar adical anion intermediate. [12] Thec omputed S 0 !S 1 vertical excitation energies at the four S 0 reactant minima are nearly identical, 63.5 kcal mol À1 for AD and BD,and 63.7 kcal mol À1 for AU and BU.They are very close to the experimentally measured absorption band maximum of 447 nm [63.7 kcal mol À1 ] [25] and previous QM/ MM results. [33] TheS 1 state is as pectroscopically bright state and mainly corresponds to ao ne-electron excitation from HOMO to LUMO (see bottom-left panel of Figure 3).…”

“…In 2013, Larsen and co-workers employed time-resolved transient absorption spectroscopy to investigate the primary and secondary photodynamics of the dark-(D 447 )a nd lightadapted (S 390 )s tates of the type-II LOVd omain of YtvA photoreceptors from Bacillus subtilis bacterium. [25] They found that the primary photodynamics (100 fs to 10 ns) of the dark-adapted YtvA is qualitatively similar to that of other type-I LOVdomains and produces ahigh triplet yield (78 %), while the secondary photodynamics (10 ns to 100 ms) is inhomogeneous with three co-evolving triplet states.T he photoexcitation of the light-adapted S 390 state was found to trigger photodissociation followed by ultrafast recombination, resulting in al ow overall dissociation yield. However, many mechanistic details remain open that are required for an in-depth understanding of the photoresponse mechanism of YtvA LOVphotoreceptors.…”

Activation of the General Stress Response of Bacillus subtilis by Visible Light

“…Photon absorption by the isoalloxazine ring of the FMN prosthetic group of YtvA generates, via the sequential involvement of electronically excited singlet and triplet states, a covalent Cys‐FMN adduct structure at the microsecond timescale with a quantum yield of ~0.45 . This covalent adduct structure causes deformation of the isoalloxazine ring and presumably flipping of a key Gln sidechain in the fifth β‐strand of the LOV domain of YtvA .…”

Photoreceptors in Chemotrophic Prokaryotes: The Case of Acinetobacter spp. Revisited

“…Strikingly, such a branching reaction is not observable in the BLUF domain of AppA , which may be related to the importance of a long lifetime of its signaling state. Significantly, absence of branching activity is also observed in YtvA, a slow‐recovering LOV domain that activates the general stress response in B. subtilis . Prior to photoactivation, the flavin chromophore of BLUF domains, like those in LOV domains, have to be in the fully oxidized form, which absorb maximally at 450 nm.…”

“…This reversible “H” transfer allows a key glutamine residue from the β ‐5 strand of the β ‐sheet of the protein to “flip” ( i.e . rotate 180 degrees); the correct positioning of a hydrophobic tryptophan side chain (W104 in AppA) is key to the success of these primary processes . This then leads to a change in the secondary structure of this β strand, because of a general weakening of the hydrogen bonds in the β ‐sheet.…”