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 (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.

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The monarch butterfly (Danaus plexippus) cryptochrome 1 (DpCry1) belongs in the class of photosensitive insect cryptochromes. Here we purified DpCry1 expressed in a bacterial host and obtained the protein with a stoichiometric amount of the flavin cofactor in the two-electron oxidized, FAD ox , form. Exposure of the purified protein to light converts the FAD ox to the FAD . flavin anion radical by intraprotein electron transfer from a Trp residue in the apoenzyme. To test whether this novel photoreduction reaction is part of the DpCry1 physiological photocycle, we mutated the Trp residue that acts as the ultimate electron donor in flavin photoreduction. The mutation, W328F, blocked the photoreduction entirely but had no measurable effect on the light-induced degradation of DpCry1 in vivo. In light of this finding and the recently published action spectrum of this class of Crys, we conclude that DpCry1 and similar insect cryptochromes do not contain flavin in the FAD ox form in vivo and that, most likely, the FAD ox --3 h FAD . photoreduction reaction is not part of the insect cryptochrome photoreaction that results in proteolytic degradation of the photopigment.Cryptochromes are blue-light photoreceptors that control growth and development in plants and the circadian clock in animals (1-3). They have sequence and structural similarities to DNA photolyases that repair far UV-induced DNA damage using blue light as a co-substrate. Photolyases contain two-electron reduced deprotonated FAD (FADH Ϫ ) as the catalytic cofactor and methenyltetrahydrofolate (MTHF) 3 or, in rare cases, 8-deazaflavin as a photoantenna for gathering photons and transferring excitation energy to FADH Ϫ to initiate catalysis (2). Cryptochromes from plant and animal sources, as well, contain FAD and are known or presumed to contain MTHF (4 -6). However, the functional redox state of flavin in cryptochromes is unknown with certainty, and there is no evidence that the flavin cofactor in cryptochrome functions as a redox catalyst (1, 3).Arabidopsis thaliana cryptochrome 1 (AtCry1), which was the first cryptochrome to be discovered (7), has been the most extensively investigated. The AtCry1, as well as the related AtCry2, can be purified as recombinant proteins expressed in Escherichia coli or baculovirus/insect cell expression systems (4,5,8). The photopigments purified from these expression systems contain trace amounts of MTHF and stoichiometric flavin cofactor in the two-electron oxidized, FAD ox , form. A number of interesting observations have been made with Arabidopsis cryptochromes, in particular with AtCry1, which may be relevant to their functions. First, AtCry1 binds ATP in the cavity containing the flavin cofactor (8 -10). Second, AtCry1 exhibits an autophosphorylating kinase activity (9 -11). Third, the FAD ox of AtCry1 is photoreduced to the FADH ⅐ neutral radical by intraprotein electron transfer both in vitro and in vivo (4, 12). Fourth, this intraprotein electron transfer causes a conformational change in the C-terminal tail of At...

Cryptochromes (CRYs) are blue-light photoreceptors with known or presumed functions in light-dependent and light-independent gene regulation in plants and animals. Although the photochemistry of plant CRYs has been studied in some detail, the photochemical behavior of animal cryptochromes remains poorly defined in part because it has been difficult to purify animal CRYs with their flavin cofactors. Here we describe the purification of type 4 CRYs of zebrafish and chicken as recombinant proteins with full flavin complement and compare the spectroscopic properties of type 4 and type 1 CRYs. In addition, we analyzed photoinduced proteolytic degradation of both types of CRYs in vivo in heterologous systems. We find that even though both types of CRYs contain stoichiometric flavin, type 1 CRY is proteolytically degraded by a light-initiated reaction in Drosophila S2, zebrafish Z3, and human HEK293T cell lines, but zebrafish CRY4 (type 4) is not. In vivo degradation of type 1 CRYs does not require continuous illumination, and a single light flash of 1 ms duration leads to degradation of about 80% of Drosophila CRY in 60 min. Finally, we demonstrate that in contrast to animal type 2 CRYs and Arabidopsis CRY1 neither insect type 1 nor type 4 CRYs have autokinase activities.

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It has recently been realized that animal cryptochromes (CRYs) fall into two broad groups. Type 1 CRYs, the prototype of which is the Drosophila CRY, that is known to be a circadian photoreceptor. Type 2 CRYs, the prototypes of which are human CRY 1 and CRY 2, are known to function as core clock proteins. The mechanism of photosignaling by the Type 1 CRYs is not well understood. We recently reported that the flavin cofactor of the Type 1 CRY of the monarch butterfly may be in the form of flavin anion radical, FAD . , in vivo. Here we describe the purification and characterization of wild-type and mutant forms of Type 1 CRYs from fruit fly, butterfly, mosquito, and silk moth. Cryptochromes from all four sources contain FAD ox when purified, and the flavin is readily reduced to FAD . by light. Interestingly, mutations that block photoreduction in vitro do not affect the photoreceptor activities of these CRYs, but mutations that reduce the stability of FAD . in vitro abolish the photoreceptor function of Type 1 CRYs in vivo. Collectively, our data provide strong evidence for functional similarities of Type 1 CRYs across insect species and further support the proposal that FAD . represents the ground state and not the excited state of the flavin cofactor in Type 1 CRYs.Cryptochromes are photolyase-related flavoproteins that play important roles in regulating the circadian clock in animals and growth and development in plants (1-3). The mechanism of photosignaling by animal cryptochromes is not known. Previously, it was thought that CRYs 3 in Drosophila and other insects function as circadian photoreceptors and in mouse and other vertebrates function as core components of the molecular clock (4). Recently, this view was revised when it was realized that some insects such as the honeybee possess only a mammalian CRY-like cryptochrome and others such as the monarch butterfly possess both Drosophila CRY-like and mammalian CRY-like cryptochromes (5, 6). It was proposed that Drosophila-like CRYs should be referred to as Type 1 CRYs and the mammalian-like CRYs should be referred to as Type 2 CRYs (6). Furthermore, it was found that all Type 1 CRYs tested were subject to light-induced proteolysis in Schneider 2 (S2) cells and, hence, were considered to function as circadian photoreceptors in a manner analogous to DmCRY (6). Similarly, it was shown that insect Type 2 CRYs, like the mammalian CRYs, functioned as core clock proteins with no demonstrable photoreceptor activity (6).We are interested in the photoreceptor function of CRY and specifically in the cryptochrome photocycle. Type 1 CRYs are well suited for this purpose because their photoinitiated proteolysis constitutes a convenient functional assay (7-9). Two recent studies reported that Type 1 CRYs from Drosophila melanogaster and the monarch butterfly (Danaus plexippus), purified as recombinant proteins, contained near-stoichiometric amounts of flavin in the two-electron oxidized, FAD ox , form. Exposure of these CRYs to light reduced the flavin to the flavin anion ...

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.

The femtosecond to nanosecond dynamics of the all-trans β-carotene carotenoid dissolved in 3-methylpentane is characterized and dissected with excitation-wavelength and temperature-dependent ultrafast dispersed transient absorption signals. The kinetics measured after red-edge (490 nm) and blue-edge (400 nm) excitation were contrasted under fluid solvent (298 K) and rigid glass (77 K) conditions. In all four measured data sets, the S* population kinetics was resolved prompting the development of a modified multicompartment model. The temperature-dependent and excitation wavelength-dependent S* quantum yield is ascribed to a competition of population surmounting a weak (55 cm(-1)) energy barrier on the S(2) state to favor S(1) generation and rapid internal conversion that favors S* generation. When cooled from room temperature to 77 K, the S* decay time scale shifted significantly from 30 to 400 ps, which is ascribed to small-scale structural relaxation with a 115 cm(-1) energy barrier. For the first time under low-energy excitation conditions, the triplet state is observed and confirmed to not originate from S* or S(1), but from S(2). The interconnectivity of the S* and S(1) populations is discussed, and no observed population flow is resolved between S* and S(1). Comparison of samples obtained from different laboratories with different purity levels demonstrates that sample contamination is not the primary origin of the S* state.