Dynamic Determination of the Functional State in Photolyase and the Implication for Cryptochrome

Dynamic determination of the functional state in photolyase and the implication for cryptochrome

Zheyun Liu a,b, Meng Zhanga,b,c, Xunmin Guo a,b, Chuang Tan a,b,d,JiangLia,b,LijuanWanga,b, Aziz Sancare,1, and Dongping Zhong a,b,c,d,f,1

Departments of aPhysics and bChemistry and Biochemistry, and Programs of cBiophysics, dChemical Physics, and fBiochemistry, The Ohio State University, Columbus, OH 43210; and eDepartment of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599

Contributed by Aziz Sancar, June 28, 2013 (sent for review May 26, 2013) – The flavin adenine dinucleotide cofactor has an unusual bent of photolyase by donating an electron from its anionic form (FAD – configuration in photolyase and cryptochrome, and such a folded in insect or FADH in plant) to a putative substrate that induces structure may have a functional role in initial photochemistry. Using a local electrostatic variation to cause conformation changes for femtosecond spectroscopy, we report here our systematic charac- signaling. Both models require electron transfer (ET) at the active terization of cyclic intramolecular electron transfer (ET) dynamics site to induce electrostatic changes for signaling. Similar to the between the flavin and adenine moieties of flavin adenine di- pyrimidine dimer, the Ade moiety near the Lf ring could also be nucleotide in four redox forms of the oxidized, neutral, and anionic an oxidant or a reductant. Thus, it is necessary to know the role of semiquinone, and anionic hydroquinone states. By comparing wild- the Ade moiety in initial photochemistry of FAD in cryptochrome type and mutant enzymes, we have determined that the excited to understand the mechanism of cryptochrome signaling. neutral oxidized and semiquinone states absorb an electron from Here, we use Escherichia coli photolyase as a model system to the adenine moiety in 19 and 135 ps, whereas the excited anionic systematically study the dynamics of the excited cofactor in semiquinone and hydroquinone states donate an electron to the four different redox forms. Using site-directed mutagenesis, we adeninemoietyin12psand2ns,respectively.AllbackETdynamics replaced all neighboring potential electron donor or acceptor occur ultrafast within 100 ps. These four ET dynamics dictate that only amino acids to leave FAD in an environment conducive to for- the anionic hydroquinone flavin can be the functional state in photo- mation of one of the four redox states. Strikingly, we observed lyaseduetotheslowerETdynamics(2ns)withtheadeninemoiety that, in all four redox states, the excited Lf proceeds to intra- BIOPHYSICS AND

and a faster ET dynamics (250 ps) with the substrate, whereas the molecular ET reactions with the Ade moiety. With femtosecond COMPUTATIONAL BIOLOGY intervening adenine moiety mediates electron tunneling for repair of resolution, we followed the entire cyclic ET dynamics and de- damaged DNA. Assuming ET as the universal mechanism for photo- termined all reaction times of wild-type and mutant forms of the lyase and cryptochrome, these results imply anionic ﬂavin as the more enzyme to reveal the molecular origin of the active state of ﬂavin attractive form of the cofactor in the active state in cryptochrome to in photolyase. With the semiclassical Marcus ET theory, we induce charge relocation to cause an electrostatic variation in the active further evaluated the driving force and reorganization energy

site and then lead to a local conformation change to initiate signaling. of every ET step in the photoinduced redox cycle to understand CHEMISTRY the key factors that control these ET dynamics. These observa- flavin functional state | intracofactor electron transfer | adenine tions may imply a possible active state among the four redox electron acceptor | adenine electron donor | femtosecond dynamics forms in cryptochrome. Results and Discussion he photolyase–cryptochrome superfamily is a class of fla- fl Photoreduction-Like ET from Adenine to Neutral Oxidized (Lf) and Tvoproteins that use avin adenine dinucleotide (FAD) as the • fl cofactor. Photolyase repairs damaged DNA (1–5), and crypto- Semiquinoid (LfH ) Lumi avins. As reported in the preceding paper (16), we have shown that the excited FAD in photolyase is chrome controls a variety of biological functions such as regulating readily quenched by the surrounding tryptophan residues, mainly plant growth, synchronizing circadian rhythms, and sensing di- W382 with a minor contribution from W384, and that the ET rection as a magnetoreceptor (6–10). Strikingly, the FAD cofactor dynamics from W382 to FAD* occurs ultrafast in 0.8 ps. By in the superfamily adopts a unique bent U-shape configuration replacing W382 and W384 to a redox inert phenylalanine (W382F/ with a close distance between its lumiflavin (Lf) and adenine (Ade) W384F) using site-directed mutagenesis, we abolished all possible moieties (Fig. 1A). The cofactor could exist in four different redox – ET between FAD* and the neighboring aromatic residues and forms (Fig. 1B): oxidized (FAD), anionic semiquinone (FAD ), • – observed a dominant decay of FAD* in 19 ps (an average time of neutral semiquinone (FADH ), and anionic hydroquinone (FADH ). – a stretched exponential decay with τ = 18 ps and β = 0.92) as shown In photolyase, the active state in vivo is FADH . We have recently −1 in Fig. 2A (kFET ) with a probing wavelength at 800 nm. The showed that the intervening Ade moiety mediates electron tun- observed stretched behavior reflects a heterogeneous quenching neling from the Lf moiety to substrate in DNA repair (5). Because dynamics, resulting from the coupling of ET with the active-site the photolyase substrate, the pyrimidine dimer, could be either solvation on the similar timescales (17). The dynamics in 19 ps an oxidant (electron acceptor) or a reductant (electron donor), reflects the intramolecular ET from the Ade to Lf moieties to a fundamental mechanistic question is why photolyase adopts + – – form a charge-separated pair of Ade +Lf . Tuning the probe FADH as the active state rather than the other three redox wavelengths to shorter than 700 nm to search for the maximum forms, and if an anionic flavin is required to donate an electron, – why not FAD , which could be easily reduced from FAD? fl In cryptochrome, the active state of the avin cofactor in vivo is Author contributions: D.Z. designed research; Z.L., M.Z., X.G., C.T., J.L., L.W., and D.Z. currently under debate. Two models of cofactor photochemistry performed research; Z.L. and D.Z. analyzed data; and Z.L., A.S., and D.Z. wrote the paper. – have been proposed (11 14). One is called the photoreduction The authors declare no conflict of interest. model (11–13), which posits that the oxidized FAD is photo- • Freely available online through the PNAS open access option. reduced mainly by a conserved tryptophan triad to neutral FADH 1 – To whom correspondence may be addressed. E-mail: [email protected] or (signaling state) in plant or FAD in insect, then triggering struc- [email protected]. tural rearrangement to initiate signaling. The other model (14, 15) This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. hypothesizes that cryptochrome uses a mechanism similar to that 1073/pnas.1311077110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1311077110 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 • Thus, beside the intrinsic lifetime, the excited LfH is likely to be quenched by intramolecular ET with Ade to form a charge- + – separated pair of Ade +LfH . Taking 230 ps as the lifetime of • LfH * without ET, we derive a forward ET dynamics with Ade in • 135 ps, contributing to an overall decay of FADH * in 85 ps. + To probe the intermediate Ade , we tuned the probe wavelengths to the shorter wavelengths to detect the maximum intermediate contribution. The best probing wavelength would be the one at which the absorption coefficients of the excited and ground • states are equal, resulting in cancellation of the positive LfH * • signal by the negative partial LfH formation signal, leading to the + dominant rise and decay signal of Ade .Fig.3B shows the typical signal probed at 555 nm. We observed negative signals due to the • initial bleaching of FADH . We can regroup all three signals • + • of LfH *, Ade ,andLfH into two dynamic types of transients • (SI Text): one represents the summation of two parts (LfH * • and LfH ) with an excited-state decay time of 100 ps and its amplitude is proportional to the difference of absorption coefficients • between the two parts. Because LfH has a larger absorption co- • • efficient (eLfH * < eLfH ), the signal flips and shows as a negative rise (Fig. 3B). The second-type transient reflects the summation of two + • + parts (Ade and LfH ) with a dynamic pattern of Ade in a rise and

Fig. 1. (A) Configuration of the FAD cofactor with four critical residues (N378, E363, W382, and W384 in green) in E. coli photolyase. The lumiflavin (Lf) (orange) and adenine (Ade) (cyan) moieties adopt an unusual bent configuration to ensure intramolecular ET within the cofactor. The N and E residues mutated to stabilize the FAD– state and the two W residues mu- • tated to leave FAD and FADH in a redox-inert environment are indicated. (B) The four redox states of FAD and their corresponding absorption spectra.

+ contribution of the putative Ade intermediate, we show two typical transients in Fig. 2 B and C probed at 630 and 580 nm, + respectively. We observed the formation of Ade in 19 ps and decay in 100 ps (see all data analyses thereafter in SI Text). The −1 decay dynamics reflects the charge recombination process (kBET ) and leads to the completion of the redox cycle. As discussed in the preceding paper (16), such ET dynamics between the Lf and Ade moieties is favorable by negative free-energy changes. Similarly, we prepared the W382F mutant in the semiquinone • state (FADH ) to eliminate the dominant electron donor of W382. Without this tryptophan in proximity, we observed a • dominant decay of FADH *in85ps(τ = 82 ps and β = 0.93) probed at 800 nm (Fig. 3A), which is similar to the previously reported 80 ps (18) that was attributed to the intrinsic lifetime of • • FADH *. In fact, the lifetime of the excited FMNH *infla- vodoxin is about 230 ps (19), which is nearly three times longer • than that of FADH * observed here. Using the reduction potentials of +1.90 V vs. normal hydrogen electrode (NHE) for Fig. 2. Femtosecond-resolved intramolecular ET dynamics between the excited oxidized Lf and Ade moieties. (A–C) Normalized transient-absorp- adenine (20) and of +0.02 V vs. NHE in photolyase for neutral • ← • tion signals of the W382F/W384F mutant in the oxidized state probed at 800, semiquinoid LfH (21), with the S1 S0 transition of FADH at 630, and 580 nm, respectively, with the decomposed dynamics of the re- + 650 nm (1.91 eV) we find that the ET reaction from Ade to actant (Lf*) and intermediate (Ade ). Inset shows the derived intramolecular • LfH * has a favorable, negative free-energy change of −0.03 eV. ET mechanism between the oxidized Lf* and Ade moieties.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1311077110 Liu et al. Downloaded by guest on October 1, 2021 – ET from Anionic Semiquinoid Lumiﬂavin (Lf ) to Adenine. In photo- – lyase, FAD cannot be stabilized and is readily converted to • FADH through proton transfer from the neighboring residues or trapped water molecules in the active site. However, in type 1 – insect cryptochromes, the ﬂavin cofactor can stay in FAD in vitro under anaerobic condition and this anionic semiquinone was also proposed to be the active state in vivo (14, 15). By examining the sequence alignment and X-ray structures (25, 26) of these two proteins, the key difference is one residue near the N5 atom of the Lf moiety, N378 in E. coli photolyase and C416 in Drosophila cryptochrome. Through structured water molecules, the N378 is connected to a surface-exposed E363 in the photolyase but C416 is connected to the hydrophobic L401 in the cryptochrome. Thus, we prepared a double-position photolyase mutant E363L/N378C to mimic the critical position near the N5 atom in the cryptochrome. With a higher pH 9 and in the presence of the thymine dimer substrate at the active site to push water molecules out of the pocket to reduce local proton donors, we were able to suc- – cessfully stabilize FAD in the mutant for more than several hours under anaerobic condition. Fig. 4 shows the absorption transients – of excited FAD probed at three wavelengths. At 650 nm (Fig. 4A), the transient shows a decay dynamics in 12 ps (τ = 12 ps and β = 0.97) without any fast component or long plateau. We also did not observe any measurable thymine dimer repair and thus ex- – clude ET from FAD * to the dimer substrate (SI Text). – The radical Lf * probably has a lifetime in hundreds of picoseconds as observed in insect cryptochrome (15), also similar to the • lifetime of the radical LfH * (19). Thus, the observed dynamics in BIOPHYSICS AND –*

12 ps must result from an intramolecular ET from Lf to Ade to COMPUTATIONAL BIOLOGY – form the Lf+Ade pair. Such an ET reaction also has a favorable driving force (ΔG0 = −0.28 eV) with the reduction potentials of – – Ade/Ade and Lf/Lf to be −2.5 and −0.3 V vs. NHE (20, 27), respectively. The observed initial ultrafast decay dynamics of – FAD * in insect cryptochromes in several to tens of picoseconds, in

addition to the long lifetime component in hundreds of pico- CHEMISTRY seconds, could be from an intramolecular ET with Ade as well as the ultrafast deactivation by a butterfly bending motion through Fig. 3. Femtosecond-resolved intramolecular ET dynamics between the – a conical intersection (15, 19) due to the large plasticity of cryp- excited neutral semiquinoid Lf and Ade moieties. (A C) Normalized tran- tochrome (28). However, photolyase is relatively rigid, and thus sient-absorption signals of the W382F mutant in the neutral semiquinoid state probed at 800, 555, and 530 nm, respectively, with the decomposed the ET dynamics here shows a single exponential decay with • fi fi dynamics of two groups: one represents the excited-state (LfH *) dynamic amorede ned con guration. behavior with the amplitude proportional to the difference of absorption Similarly, we tuned the probe wavelengths to the blue side to • • + – coefficients between LfH * and LfH ; the other gives the intermediate (Ade ) probe the intermediate states of Lf and Ade and minimize the dynamic behavior with the amplitude proportional to the difference of total contribution of the excited-state decay components. Around + • absorption coefficients between Ade and LfH . Inset shows the derived 350 nm, we detected a significant intermediate signal with a rise in •* intramolecular ET mechanism between the neutral LfH and Ade moieties. 2 ps and a decay in 12 ps. The signal flips to the negative ab- ∼ – For the weak signal probed at 555 nm, a long component ( 20%) was re- sorption due to the larger ground-state Lf absorption. Strikingly, moved for clarity and this component could be from the product(s) resulting C from the excited state due to the short lifetime of 230 ps. at 348 nm (Fig. 4 ), we observed a positive component with the excited-state dynamic behavior (eLf–* > eLf–)andaflipped negative component with a rise and decay dynamic profile (eLf+ e – < e –). Clearly, the observed 2 ps dynamics reflects the decay behavior and similarly the signal flips due to the larger ab- Ade Lf fi • back ET dynamics and the intermediate signal with a slow for- sorption coef cient of FADH . mation and a fast decay appears as apparent reverse kinetics Kinetically, we observed an apparent rise in 20 ps and a decay in again. This observation is significant and explains why we did not 85 ps. Fig. 3C shows that, when the transient is probed at 530 nm, • observe any noticeable thymine dimer repair due to the ultrafast the ground-state LfH recovery in 85 ps dominates the signal. fl back ET to close redox cycle and thus prevent further electron Thus, the observed dynamics in 20 ps re ects the back ET process tunneling to damaged DNA to induce dimer splitting. Thus, in and the signal manifests as apparent reverse kinetics, leading to wild-type photolyase, the ultrafast cyclic ET dynamics determines – less accumulation of the intermediate state. Here, the charge re- that FAD cannot be the functional state even though it can donate combination in 20 ps is much faster than the charge separation in one electron. The ultrafast back ET dynamics with the intervening 135 ps with a driving force of −1.88 eV in the Marcus inverted • Ade moiety completely eliminates further electron tunneling to the region. In summary, although the neutral FAD* and FADH * dimer substrate. Also, this observation explains why photolyase uses – – states can draw an electron from a strong reductant and the dimer fully reduced FADH as the catalytic cofactor rather than FAD – substrate can be repaired by a strong oxidant (22) by donating an even though FAD can be readily reduced from the oxidized FAD. electron to induce cationic dimer splitting, the ultrafast cyclic ET – dynamics with the Ade moiety in the mutants reported here or with ET from Anionic Hydroquinoid Lumiflavin (LfH ) to Adenine. Pre- – the neighboring tryptophans in the wild type (23, 24) exclude these viously, we reported the total lifetime of 1.3 ns for FADH * (2). two neutral redox states as the functional state in photolyase. Because the free-energy change ΔG0 for ET from fully reduced

Liu et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 mechanism with two tunneling steps from the cofactor to adenine and then to dimer substrate. Due to the favorable driving force, the electron directly tunnels from the cofactor to dimer substrate and on the tunneling pathway the intervening Ade moiety mediates the ET dynamics to speed up the ET reaction in the ﬁrst step of repair (5).

Unusual Bent Configuration, Intrinsic ET, and Unique Functional State. With various mutations, we have found that the intramolecular ET between the flavin and the Ade moiety always occurs with the bent configuration in all four different redox states of photolyase and cryptochrome. The bent flavin structure in the active site is unusual among all flavoproteins. In other flavoproteins, the flavin cofactor mostly is in an open, stretched configuration, and if any, the ET dynamics would be longer than the lifetime due to the long separation distance. We have found that the Ade moiety mediates the initial ET dynamics in repair of damaged DNA using this unusual bent structure (5, 29). Currently, it is not known whether the bent structure has a functional role in cryptochrome. If the – – active state is FAD in type 1 insect cryptochromes or FADH in

Fig. 4. Femtosecond-resolved intramolecular ET dynamics between the excited anionic semiquinoid Lf and Ade moieties. (A–C) Normalized transient-absorption signals of the E363L/N378C mutant in the anionic semiquinoid state probed at 650, 350, and 348 nm, respectively, with the decomposed dynamics of two groups: one exhibits the excited-state (Lf–*) dynamic behavior with the amplitude proportional to the difference of – – absorption coefﬁcients between Lf * and Lf ; the other has the intermediate – (Lf or Ade ) dynamic behavior with the amplitude proportional to the dif- – – ference of absorption coefﬁcients between (Lf+Ade ) and Lf . Inset shows – the derived intramolecular ET mechanism between the anionic Lf * and Ade moieties.

– LfH * to adenine is about +0.04 eV (5, 21), the ET dynamics could occur on a long timescale. We observed that the fluores- cence and absorption transients all show the excited-state decay dynamics in 1.3 ns (Fig. 5A, τ = 1.2 ns and β = 0.90). Similarly, we needed to tune the probe wavelengths to maximize the intermediate absorption and minimize the contributions of excited- state dynamic behaviors. According to our previous studies (4, 5), at around 270 nm both the excited and ground states have similar absorption coefficients. Fig. 5 B and C show the transients probed • around 270 nm, revealing that the intermediate LfH signal is e •+e – > e –+e positive ( LfH Ade LfH Ade) and dominant. Similarly, we Fig. 5. Femtosecond-resolved intramolecular ET dynamics between the observed an apparent reverse kinetics with a rise in 25 ps and excited anionic hydroquinoid Lf and Ade moieties. (A–C) Normalized tran- a decay in 1.3 ns. With the N378C mutant, we reported the life- sient-absorption signals in the anionic hydroquinoid state probed at 800, –* 270, and 269 nm with the decomposed dynamics of two groups: one rep- time of FADH as 3.6 ns (4) and taking this value as the lifetime – resents the excited-state (LfH *) dynamic behavior with the amplitude pro- without ET with the Ade moiety, we obtain the forward ET time –* fl portional to the difference of absorption coefficients between LfH and as 2 ns. Thus, the rise dynamics in 25 ps re ects the back ET and LfH–; the other reflects the intermediate (LfH• or Ade–) dynamic behavior this process is ultrafast, much faster than the forward ET. This with the amplitude proportional to the difference of absorption coefficients • – – observation is significant and indicated that the ET from the co- between (LfH +Ade ) and (LfH +Ade). Inset shows the derived intramolecular – factor to the dimer substrate in 250 ps does not follow the hopping ET mechanism between the anionic LfH * and Ade moieties.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1311077110 Liu et al. Downloaded by guest on October 1, 2021 plant cryptochrome, then the intramolecular ET dynamics with treated in the preceding paper (16) and evaluate the driving the Ade moiety could be significant due to the charge relocation forces (ΔG0) and reorganization energies (λ) for the ET reac- to cause an electrostatic change, even though the back ET could tions of the four redox states. Because no significant conforma- be ultrafast, and such a sudden variation could induce local con- tion variation in the active site for different redox states is formation changes to form the initial signaling state. Conversely, observed (31), we assume that all ET reactions have the similar if the active state is FAD, the ET dynamics in the wild type of electronic coupling constant of J = 12 meV as reported for the cryptochrome is ultrafast at about 1 ps with the neighboring oxidized state (16). With assumption that the reorganization tryptophan(s) and the charge recombination is in tens of pico- energy of the back ET is larger than that of the forward ET, we seconds (15). Such ultrafast change in electrostatics could be solved the driving force and reorganization energy of each ET – similar to the variation induced by the intramolecular ET of FAD * step and the results are shown in Fig. 6B with a 2D contour – or FADH *. Thus, the unusual bent configuration assures an plot. The driving forces of all forward ET fall in the region “intrinsic” intramolecular ET within the cofactor to induce a large between +0.04 and −0.28 eV, whereas the corresponding back electrostatic variation for local conformation changes in crypto- ET is in the range from −1.88 to −2.52 eV. The reorganization chrome, which may imply its functional role. energy of the forward ET varies from 0.88 to 1.10 eV, whereas We believe the findings reported here explain why the active the back ET acquires a larger value from 1.11 to 1.64 eV. These – state of flavin in photolyase is FADH . With the unusual bent values are consistent with our previous findings about the re- configuration, the intrinsic ET dynamics determines the only organization energy of flavin-involved ET in photolyase (5), – – choice of the active state to be FADH , not FAD , due to the which is mainly contributed by the distortion of the flavin co- much slower intramolecular ET dynamics within the cofactor in factor during ET (close to 1 eV). All forward ET steps fall in the the former (2 ns) than in the latter (12 ps), although both anionic Marcus normal region due to their small driving forces and all of redox states could donate one electron to the dimer substrate. the back ET processes are in the Marcus inverted region. Note • With the neutral redox states of FAD and FADH , the ET dy- that the back ET dynamics of the anionic cofactors (2 and 4 in namics are ultrafast with the neighboring aromatic tryptophan(s) Fig. 6B) have noticeably larger reorganization energies than even though the dimer substrate could donate one electron to those with the neutral flavins probably because different high- the neutral cofactor, but the ET dynamics is not favorable, being frequency vibrational energy is involved in different back ETs. much slower than those with the tryptophans or the Ade moiety. Overall, the ET dynamics are controlled by both free-energy Thus, the only active state for photolyase is anionic hydroqui- change and reorganization energy as shown in Fig. 6B. The

– BIOPHYSICS AND none FADH with an unusual, bent conﬁguration due to the active site of photolyase modulates both factors to control the unique dynamics of the slower intramolecular ET (2 ns) in the ET dynamics of charge separation and recombination or COMPUTATIONAL BIOLOGY cofactor and the faster intermolecular ET (250 ps) with the di- charge relocations in every redox state. mer substrate (4). These intrinsic intramolecular cyclic ET dynamics in the four redox states are summarized in Fig. 6A. Conclusion We reported here our direct observation of intramolecular ET Energetics of ET in Photolyase Analyzed by Marcus Theory. The in- between the Lf and Ade moieties with an unusual bent conﬁgu-

trinsic intramolecular ET dynamics in the unusual bent cofactor ration of the flavin cofactor in photolyase in four different redox CHEMISTRY configuration with four different redox states all follow a single states using femtosecond spectroscopy and site-direct mutagen- exponential decay with a slightly stretched behavior (β = 0.90– esis. Upon blue-light excitation, the neutral oxidized and semi- 0.97) due to the compact juxtaposition of the flavin and Ade quinone lumiflavins can be photoreduced by accepting an electron moieties in FAD. Thus, these ET dynamics are weakly coupled from the Ade moiety (or neighboring aromatic tryptophans), with local protein relaxations. With the cyclic forward and back while the anionic semiquinone and hydroquinone lumiflavins can ET rates, we can use the semiempirical Marcus ET theory (30) as reduce the Ade moiety by donating an electron. After the initial

Fig. 6. Summary of the molecular mechanisms and dynamics of cyclic intramolecular ET between the Lf and Ade moieties of photolyase in the four different redox states and their dependence on driving forces and reorganization energies. (A) Reaction times and mechanisms of the cyclic ET between the Lf and Ade moieties in all four redox states. (B) Two-dimensional contour plot of the ET times relative to free energy (ΔG0) and reorganization energy (λ) for all electron tunneling steps. All forward ET reactions are in the Marcus normal region (−ΔG0 < λ), whereas all back ET steps are in the Marcus inverted region (−ΔG0 > λ).

Liu et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 charge separation or relocation, all back ET dynamics occur ul- Materials and Methods trafast in less than 100 ps to close the photoinduced redox cycle. Photolyase Mutants and Their Redox States. The preparation of His-tag fused Strikingly, in contrast to the oxidized state, all other three back ET E. coli photolyase E109A mutant (EcPL) has been described before (32). Based dynamics are much faster than their forward ET processes, on this template, we mutated two tryptophans of W382 and W384 near the flavin and prepared this double mutant in the oxidized form (FAD). For the leading to less accumulation of the intermediate state. To capture – fi anionic semiquinone (FAD ), we mutated two critical positions in EcPL of E363L the intermediate states, it is necessary to nd an appropriate and N378C. After purification, we obtained the mutant protein in completely probing wavelength to cancel out the contributions from both the oxidized state. Before ultrafast experiments, the mutant enzyme of a concen- excited state (positive signal) and ground state (negative signal), tration of 100 μM was exchanged into a basic reaction buffer at pH 9 with · leaving the weak intermediate signal dominant. 50 mM Tris HCl, 300 mM NaCl, 1 mM EDTA, 20 mM DTT, 1 mM oligo-(dT)15 The intramolecular ET dynamics in the four redox states with containing cyclobutane thymine dimers, and 50% (vol/vol) glycerol. After fi purge with argon and irradiation with UV light at 365 nm (UVP; 8 W), the flavin the bent cofactor con guration reveal the molecular origin of the – cofactor is stabilized at the FAD state under anaerobic conditions. The neutral active state in photolyase and imply a universal ET model for • semiquinone (FADH ) EcPL was prepared by mutation of W382F in EcPL and both photolyase and cryptochrome. To repair damaged DNA in the anionic hydroquinone (FADH–) EcPL was stabilized under anaerobic con- photolyase, the ET must be from the anionic flavin cofactor and ditions after purge with argon and subsequent photoreduction. the intramolecular ET dynamics unambiguously reveal that only – – the FADH as the active state rather than FAD due to the in- Femtosecond Absorption Spectroscopy. All of the femtosecond-resolved trinsically slower ET (2 ns) in the former and faster ET (12 ps) in measurements were carried out using the transient-absorption method. The experimental layout has been detailed previously (24). Enzyme preparations the latter, allowing a feasible, relatively fast, ET (250 ps) to the – – with oxidized (FAD) and anionic semiquinone (FAD ) flavin were excited at damaged-DNA substrate from FADH with the intervening Ade • 480 nm. For enzyme with neutral semiquinone (FADH ), the pump wave- moiety in the middle to mediate such initial electron tunneling – • length was set at 640 nm. For the anionic hydroquinone (FADH ) form of the for repair. In cryptochrome, either neutral FAD and FADH or enzyme, we used 400 nm as the excitation wavelength. The probe wave- – – anionic FAD and FADH can proceed to an ET dynamics upon lengths were tuned to cover a wide range of wavelengths from 800 to blue-light excitation. For the former, the ET with the neigh- 260 nm. The instrument time resolution is about 250 fs and all of the boring aromatic tryptophans occur in 1 and 45 ps or with the Ade experiments were done at the magic angle (54.7°). Samples were kept stirring during irradiation to avoid heating and photobleaching. Experiments with moiety in 19 and 135 ps, and for the latter, the ET with the Ade • the neutral FAD and FADH states were carried out under aerobic conditions, – – moiety occur in 12 ps and 2 ns, respectively. All back ET dy- whereas those with the anionic FAD and FADH states were executed under namics occur within 100 ps. Such ET dynamics induce an elec- anaerobic conditions. All experiments were performed in quartz cuvettes trostatic variation in the active site, leading to local conformation with a 5-mm optical length except that the FADH– experiments probed at 270 changes to form the initial signaling state. A unified ET mech- and 269 nm were carried out in quartz cuvettes with a 1-mm optical length. anism for both photolyase and cryptochrome would imply that an anionic redox form is more attractive as a functional state in ACKNOWLEDGMENTS. This work is supported in part by National Institutes of Health Grants GM074813 and GM31082, the Camille Dreyfus Teacher– cryptochrome. Further studies are needed, however, to understand Scholar (to D.Z.), the American Heart Association fellowship (to Z.L.), and the signaling mechanism(s) of photosensory cryptochrome. The Ohio State University Pelotonia fellowship (to C.T. and J.L.).

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