Martin Byrdin

The Cryptochromes: Blue Light Photoreceptors in Plants and Animals

Cryptochromes are flavoprotein photoreceptors first identified in Arabidopsis thaliana, where they play key roles in growth and development. Subsequently identified in prokaryotes, archaea, and many eukaryotes, cryptochromes function in the animal circadian clock and are proposed as magnetoreceptors in migratory birds. Cryptochromes are closely structurally related to photolyases, evolutionarily ancient flavoproteins that catalyze light-dependent DNA repair. Here, we review the structural, photochemical, and molecular properties of cry-DASH, plant, and animal cryptochromes in relation to biological signaling mechanisms and uncover common features that may contribute to better understanding the function of cryptochromes in diverse systems including in man.

Reaction mechanisms of DNA photolyase

DNA photolyase uses visible light and a fully reduced flavin cofactor FADH(-) to repair major UV-induced lesions in DNA, the cyclobutane pyrimidine dimers (CPDs). Electron transfer from photoexcited FADH(-) to CPD, splitting of the two intradimer bonds, and back electron transfer to the transiently formed flavin radical FADH° occur in overall 1ns. Whereas the kinetics of FADH° was resolved, the DNA-based intermediates escaped unambiguous detection yet. Another light reaction, named photoactivation, reduces catalytically inactive FADH° to FADH(-) without implication of DNA. It involves electron hopping along a chain of three tryptophan residues in 30ps, as elucidated in detail by transient absorption spectroscopy. The same triple tryptophan chain is found in cryptochrome blue-light photoreceptors and may be involved in their primary photoreaction.

Dissection of the triple tryptophan electron transfer chain in Escherichia coli DNA photolyase: Trp382 is the primary donor in photoactivation

In Escherichia coli photolyase, excitation of the FAD cofactor in its semireduced radical state (FADH•) induces an electron transfer over ≈15 Å from tryptophan W306 to the flavin. It has been suggested that two additional tryptophans are involved in an electron transfer chain FADH• ← W382 ← W359 ← W306. To test this hypothesis, we have mutated W382 into redox inert phenylalanine. Ultrafast transient absorption studies showed that, in WT photolyase, excited FADH• decayed with a time constant τ ≈ 26 ps to fully reduced flavin and a tryptophan cation radical. In W382F mutant photolyase, the excited flavin was much longer lived (τ ≈ 80 ps), and no significant amount of product was detected. We conclude that, in WT photolyase, excited FADH• is quenched by electron transfer from W382. On a millisecond scale, a product state with extremely low yield (≈0.5% of WT) was detected in W382F mutant photolyase. Its spectral and kinetic features were similar to the fully reduced flavin/neutral tryptophan radical state in WT photolyase. We suggest that, in W382F mutant photolyase, excited FADH• is reduced by W359 at a rate that competes only poorly with the intrinsic decay of excited FADH• (τ ≈ 80 ps), explaining the low product yield. Subsequently, the W359 cation radical is reduced by W306. The rate constants of electron transfer from W382 to excited FADH• in WT and from W359 to excited FADH• in W382F mutant photolyase were estimated and related to the donor–acceptor distances.

What makes the difference between a cryptochrome and DNA photolyase? A spectroelectrochemical comparison of the flavin redox transitions

Cryptochromes and DNA photolyases are highly homologous flavoproteins that accomplish completely different tasks. While plant cryptochrome1 functions as blue light photoreceptor that triggers various morphogenic reactions, photolyases repair UV-induced DNA damages. Both enzymes share the photoactive cofactor, noncovalently bound FAD. For photolyase, the reaction mechanism involves electron transfer to the substrate from the excited-state of fully reduced flavin. For cryptochrome, photoexcitation of the oxidized flavin leads to formation of the semireduced radical FADH(*). Key parameters for the redox state of the flavin in the cell are the midpoint potentials E(1) and E(2) for the oxidized/semireduced and semireduced/fully reduced transitions, respectively. A link between cryptochrome function and its cofactors redox states has been suggested early on, but no reliable determinations of midpoint potentials have been available. Here we report spectroelectrochemical titrations of cryptochrome1 from Arabidopsis thaliana and photolyases from both E. coli and Anacystis nidulans at pH 7.4. For the cryptochrome, we obtained E(1) approximately E(2) approximately -160 mV vs NHE, strongly deviating from the photolyases where FADH(*) could not be oxidized up to 400 mV, and E(2) approximately -40 mV. Functional and evolutionary implications are discussed, highlighting the role of an asparagine-to-aspartate replacement close to N5 of the flavin.

Kinetics of cyclobutane thymine dimer splitting by DNA photolyase directly monitored in the UV

CPD photolyase uses light to repair cyclobutane pyrimidine dimers (CPDs) formed between adjacent pyrimidines in UV-irradiated DNA. The enzyme harbors an FAD cofactor in fully reduced state (FADH-). The CPD repair mechanism involves electron transfer from photoexcited FADH- to the CPD, splitting of its intradimer bonds, and electron return to restore catalytically active FADH-. The two electron transfer processes occur on time scales of 10-10 and 10-9 s, respectively. Until now, CPD splitting itself has only been poorly characterized by experiments. Using a previously unreported transient absorption setup, we succeeded in monitoring cyclobutane thymine dimer repair in the main UV absorption band of intact thymine at 266 nm. Flavin transitions that overlay DNA-based absorption changes at 266 nm were monitored independently in the visible and subtracted to obtain the true repair kinetics. Restoration of intact thymine showed a short lag and a biexponential rise with time constants of 0.2 and 1.5 ns. We assign these two time constants to splitting of the intradimer bonds (creating one intact thymine and one thymine anion radical T∘-) and electron return from T∘- to the FAD cofactor with recovery of the second thymine, respectively. Previous model studies and computer simulations yielded various CPD splitting times between < 1 ps and < 100 ns. Our experimental results should serve as a benchmark for future efforts to model enzymatic photorepair. The technique and methods developed here may be applied to monitor other photoreactions involving DNA.

Phototransformable fluorescent proteins: Future challenges

In fluorescence microscopy, the photophysical properties of the fluorescent markers play a fundamental role. The beauty of phototransformable fluorescent proteins (PTFPs) is that some of these properties can be precisely controlled by light. A wide range of PTFPs have been developed in recent years, including photoactivatable, photoconvertible and photoswitchable fluorescent proteins. These smart labels triggered a plethora of advanced fluorescence methods to scrutinize biological cells or organisms dynamically, quantitatively and with unprecedented resolution. Despite continuous improvements, PTFPs still suffer from limitations, and mechanistic questions remain as to how these proteins precisely work.

Structural evidence for a two-regime photobleaching mechanism in a reversibly switchable fluorescent protein.

Photobleaching, the irreversible photodestruction of a chromophore, severely limits the use of fluorescent proteins (FPs) in optical microscopy. Yet, the mechanisms that govern photobleaching remain poorly understood. In Reversibly Switchable Fluorescent Proteins (RSFPs), a class of FPs that can be repeatedly photoswitched between nonfluorescent and fluorescent states, photobleaching limits the achievable number of switching cycles, a process known as photofatigue. We investigated the photofatigue mechanisms in the protein IrisFP using combined X-ray crystallography, optical in crystallo spectroscopy, mass spectrometry and modeling approaches. At laser-light intensities typical of conventional wide-field fluorescence microscopy, an oxygen-dependent photobleaching pathway was evidenced. Structural modifications induced by singlet-oxygen production within the chromophore pocket revealed the oxidation of two sulfur-containing residues, Met159 and Cys171, locking the chromophore in a nonfluorescent protonated state. At laser-light intensities typical of localization-based nanoscopy (>0.1 kW/cm(2)), a completely different, oxygen-independent photobleaching pathway was found to take place. The conserved Glu212 underwent decarboxylation concomitantly with an extensive rearrangement of the H-bond network around the chromophore, and an sp(2)-to-sp(3) hybridization change of the carbon atom bridging the chromophore cyclic moieties was observed. This two-regime photobleaching mechanism is likely to be a common feature in RSFPs from Anthozoan species, which typically share high structural and sequence identity with IrisFP. In addition, our results suggest that, when such FPs are used, the illumination conditions employed in localization-based super-resolution microscopy might generate less cytotoxicity than those of standard wide-field microscopy at constant absorbed light-dose. Finally, our data will facilitate the rational design of FPs displaying enhanced photoresistance.

Additive Effect of Mutations Affecting the Rate of Phylloquinone Reoxidation and Directionality of Electron Transfer within Photosystem I

Optical pump‐probe spectroscopy in the nanosecond–microsecond timescale has been used to study the electron transfer reactions taking place within the Photosystem I reaction center of intact Chlamydomonas reinhardtii cells. The biphasic kinetics of phylloquinone (PhQ) reoxidation were investigated in double mutants that combine a mutation (PsaA‐Y696F) near the primary acceptor chlorophyll, ec3A, with those near PhQA (PsaA‐S692A, PsaA‐W697F). The PsaA‐S692A and PsaA‐W697F mutations selectively lengthened the 200 ns lifetime component observed in the wild‐type (WT). The ?20 ns component was unaltered in the single mutant, both in terms of lifetime and relative amplitude. However, both double mutants possessed a ?20 ns component (PhQB− reoxidation) with increased amplitude compared with the WT and the individual PhQA mutants. The component assigned to PhQA− reoxidation was slowed, like the individual PhQA mutants, and of lower amplitude, as observed in the single ec3A mutant. Hence, the effects of these mutations are almost entirely additive, providing strong support for the previously proposed bidirectional electron transfer model, which attributes the ?20 and ?200 ns phases to reoxidation of PhQB or PhQA, respectively. Moreover, in all the mutants investigated, it was also possible to observe an intermediate (∼180 ns) component, as previously reported for mutants of the PhQA binding pocket (Biochim. Biophys. Acta [2006] 1757, 1529–1538), which we have tentatively attributed to forward electron transfer between the iron–sulfur clusters FX and FA/B.

Serial Femtosecond Crystallography and Ultrafast Absorption Spectroscopy of the Photoswitchable Fluorescent Protein Irisfp.

Reversibly photoswitchable fluorescent proteins find growing applications in cell biology, yet mechanistic details, in particular on the ultrafast photochemical time scale, remain unknown. We employed time-resolved pump-probe absorption spectroscopy on the reversibly photoswitchable fluorescent protein IrisFP in solution to study photoswitching from the nonfluorescent (off) to the fluorescent (on) state. Evidence is provided for the existence of several intermediate states on the pico- and microsecond time scales that are attributed to chromophore isomerization and proton transfer, respectively. Kinetic modeling favors a sequential mechanism with the existence of two excited state intermediates with lifetimes of 2 and 15 ps, the second of which controls the photoswitching quantum yield. In order to support that IrisFP is suited for time-resolved experiments aiming at a structural characterization of these ps intermediates, we used serial femtosecond crystallography at an X-ray free electron laser and solved the structure of IrisFP in its on state. Sample consumption was minimized by embedding crystals in mineral grease, in which they remain photoswitchable. Our spectroscopic and structural results pave the way for time-resolved serial femtosecond crystallography aiming at characterizing the structure of ultrafast intermediates in reversibly photoswitchable fluorescent proteins.

Very Fast Product Release and Catalytic Turnover of DNA Photolyase

and enters into the binding pocket of the enzyme, thus approaching the FAD cofactor (3.1 in CPD photolyase and even 2.7 in the closely related (6–4) photolyase.) Following excitation of FAD in its fully reduced state (FADH ), an electron is transferred to the CPD, thereby initiating the splitting of the dimer, and subsequently returned to the flavin. The overall rate krepair of these photochemical repair steps is ~10 s . Despite the fast repair reaction, the catalytic turnover number of photolyase under saturating continuous light has been reported to be only in the order of 0.1 to 1 s ; this suggests that exchange of repaired DNA (product) for damaged DNA (substrate) is slow and rate limiting. While substrate binding was found to be rather fast (k1~10 m 1 s 1 [12, ), the rate of product release (k2) has not been established as yet, and this step might well be rate limiting. Interestingly, in an X-ray diffraction experiment at 100 K, the restored pyrimidines remained in the binding pocket in close proximity to the flavin throughout data collection. Here, we accessed product-forsubstrate exchange directly with a time-resolved experiment based on the well-established 14, 15] quenching of FADH fluorescence by electron transfer to a T<>T–CPD present in the binding pocket. Surprisingly, following an intense repair flash, the flavin fluorescence did not recover immediately, but rather with a time constant of ~50 ms (at 10 8C). This observation suggests that the restored thymines act as electron acceptors and hence as fluorescence quenchers of nearby excited FADH ; the hitherto unobserved 50 ms kinetics then reflect product release from the binding pocket and set an upper limit of 2 10 s 1 to the catalytic turnover number of photolyase. To verify this prediction, we re-examined the turnover number of photolyase under strong continuous laser light and a high substrate concentration. A rate of 260 s 1 was observed, more than 100 times faster than previously reported. The quenching of FADH fluorescence by the CPD has been used for titration of binding processes by applying steadystate fluorescence spectroscopy. Time-resolved fluorescence studies revealed that the fluorescence decay of FADH accelerates from 1.4 ns in the absence of substrate to 160 ps when a CPD is bound. Based on this difference in lifetime, we designed an experiment of the pump–probe principle: a strong actinic flash is applied to repair the substrate bound at the ACHTUNGTRENNUNGenzyme’s active site, and a weak probe flash at variable time delay serves to read out the fluorescence intensity and kinetics as a measure for the occupation of the substrate binding pocket. To avoid complications due to the antenna chromophore present in photolyases, we used an apophotolyase from Anacystis nidulans that was overexpressed in E. coli and is devoid of the 8-HDF antenna chromophore. The substrate was a UV-irradiated dT18 oligonucleotide that contained on average six randomly distributed CPDs (see the Supporting Information for details on enzyme and substrate preparation). Note that our substrate is heterogeneous with respect to the number and distribution of the CPDs per strand. Presence of this substrate quenched the steady-state fluorescence of FADH in photolyase by 83 % (Figure 1, inset). Time-resolved FADH fluorescence traces induced by a weak probe flash of 100 ps duration are presented in the main panel of Figure 1. In the absence of substrate (thick black trace), the fluorescence had a lifetime of 1.3 ns (thin black line; see SupScheme 1. Schematic representation of the catalytic cycle of DNA photolyase.