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The article studies the purification of type 4 cryptochromes (CRYs) of zebrafish and chicken as recombinant proteins with full flavin complement and evaluate the spectroscopic properties of type 4 and type 1 CRYs. Results revealed that in contrast to animal type 2 CRYs and Arabidopsis CRY1 neither insect type 1 nor type 4 CRYs has autokinase activities.

A systematic study of the dynamics of four redox states of the flavin cofactor in both photolyases and insect type 1 cryptochromes is reported. The unique properties of anionic flavins provide insight into the universal mechanism of electron transfer for the initial functional steps of the photolyase/cryptochrome blue-light photoreceptor family.

The primary (100 fs to 10 ns) and secondary (10 ns to 100 //s) 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 ran 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, wliich 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 1T" population (38% occupancy) with a 200 ns decay time is nonproductive in generating the S390 adduct state, a slower IIT" population (57% occupancy) exhibits a high yield (Φadduct ≈ 100%) in generating S390 and a third (5%) IIIT"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 capiUus-veneris [Kermis, J. T., et al. (2004) J. Am. Chcm. Soa 126, 4512], with a low-yield dissociation (Φdis ≈ 2.5%) reaction, wliich is due to an ultrafast recombination reaction, following photodissociation, and is absent in AcLOV2, which results in die increased photoswitching activity of the latter domain. [ABSTRACT FROM AUTHOR]

Phototropins, a class of light-activated protein kinases, are essential for several blue light responses in plants and algae, including phototropism. These proteins contain two internal light, oxygen, and voltage sensitive (LOV) domains, which bind flavin chromophores and undergo a reversible photochemical formation of a cysteinyl-flavin adduct as part of the light sensing process. While the photodynamic properties of such photosensory domains are dictated by interactions between the chromophore and surrounding protein, more distant residues can play a significant role as well. Here we explore the role of the Phe434 residue in the photosensory response of the second LOV domain of Avena sativa phototropin 1 (AsLOV2), a model photochemical system for these LOV domains. Phe434 is more than 6 Å from the FMN chromophore in AsLOV2; nevertheless, an F434Y point mutation is likely to change several structural features of the chromophore binding site, as we demonstrate using molecular dynamics simulations. Transient absorption signals spanning 15 decades in time were compared for wild-type AsLOV2 and the F434Y mutant, showing that the latter has significantly altered photodynamics, including (i) a faster intersystem crossing leading to triplet formation on a nanosecond time scale, (ii) biphasic formation of adduct-state kinetics on the microsecond time scale, and (iii) greatly accelerated ground-state recovery kinetics on a second time scale. We present mechanistic models that link these spectroscopic differences to changes in the configuration of the critical cysteine residue and in the chromophore's accessibility to solvent and oxygen according to MD trajectories and purging experiments. Taken together, these results demonstrate the importance of residues outside the chromophore-binding pocket in modulating LOV domain photodynamics. [ABSTRACT FROM AUTHOR]

We report here our systematic studies of the dynamics of four redox states of the flavin cofactor in both photolyases and insect type 1 cryptochromes. With femtosecond resolution, we observed ultrafast photoreduction of oxidized state flavin adenine dinucleotide (FAD) in subpicosecond and of neutral radical semiquinone (FADH•) in tens of picoseconds through intraprotein electron transfer mainly with a neighboring conserved tryptophan triad. Such ultrafast dynamics make these forms of flavin unlikely to be the functional states of the photolyase/cryptochrome family. In contrast, we find that upon excitation the anionic semiquinone (FAD•-) and hydroquinone (FADH-) have longer lifetimes that are compatible with high- efficiency intermolecular electron transfer reactions. In photolyases, the excited active state (FADH-*) has a long (nanosecond) lifetime optimal for DNA-repair function. In insect type 1 cryptochromes known to be blue-light photoreceptors the excited active form (FAD•-*) has complex deactivation dynamics on the time scale from a few to hundreds of picoseconds, which is believed to occur through conical intersection(s) with a flexible bending motion to modulate the functional channel. These unique properties of anionic flavins suggest a universal mechanism of electron transfer for the initial functional steps of the photolyase/cryptochrome blue-light photoreceptor family. [ABSTRACT FROM AUTHOR]