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

Abstract

The flavin adenine dinucleotide cofactor has an unusual bent configuration in photolyase and cryptochrome, and such a folded structure may have a functional role in initial photochemistry. Using femtosecond spectroscopy, we report here our systematic characterization of cyclic intramolecular electron transfer (ET) dynamics between the flavin and adenine moieties of flavin adenine dinucleotide in four redox forms of the oxidized, neutral, and anionic semiquinone, and anionic hydroquinone states. By comparing wild-type and mutant enzymes, we have determined that the excited neutral oxidized and semiquinone states absorb an electron from the adenine moiety in 19 and 135 ps, whereas the excited anionic semiquinone and hydroquinone states donate an electron to the adenine moiety in 12 ps and 2 ns, respectively. All back ET dynamics occur ultrafast within 100 ps. These four ET dynamics dictate that only the anionic hydroquinone flavin can be the functional state in photolyase due to the slower ET dynamics (2 ns) with the adenine moiety and a faster ET dynamics (250 ps) with the substrate, whereas the intervening adenine moiety mediates electron tunneling for repair of damaged DNA. Assuming ET as the universal mechanism for photolyase and cryptochrome, these results imply anionic flavin as the more attractive form of the cofactor in the active state in cryptochrome to induce charge relocation to cause an electrostatic variation in the active site and then lead to a local conformation change to initiate signaling.

The photolyase–cryptochrome superfamily is a class of flavoproteins that use flavin adenine dinucleotide (FAD) as the cofactor. Photolyase repairs damaged DNA (1–5), and cryptochrome controls a variety of biological functions such as regulating plant growth, synchronizing circadian rhythms, and sensing direction as a magnetoreceptor (6–10). Strikingly, the FAD cofactor in the superfamily adopts a unique bent U-shape configuration with a close distance between its lumiflavin (Lf) and adenine (Ade) moieties (Fig. 1A). The cofactor could exist in four different redox forms (Fig. 1B): oxidized (FAD), anionic semiquinone (FAD^–), neutral semiquinone (FADH^•), and anionic hydroquinone (FADH^–). In photolyase, the active state in vivo is FADH^–. We have recently showed that the intervening Ade moiety mediates electron tunneling from the Lf moiety to substrate in DNA repair (5). Because the photolyase substrate, the pyrimidine dimer, could be either an oxidant (electron acceptor) or a reductant (electron donor), a fundamental mechanistic question is why photolyase adopts FADH^– as the active state rather than the other three redox forms, and if an anionic flavin is required to donate an electron, why not FAD^–, which could be easily reduced from FAD?

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 mutated to leave FAD and FADH^• in a redox-inert environment are indicated. (B) The four redox states of FAD and their corresponding absorption spectra.

In cryptochrome, the active state of the flavin cofactor in vivo is currently under debate. Two models of cofactor photochemistry have been proposed (11–14). One is called the photoreduction model (11–13), which posits that the oxidized FAD is photoreduced mainly by a conserved tryptophan triad to neutral FADH^• (signaling state) in plant or FAD^– in insect, then triggering structural rearrangement to initiate signaling. The other model (14, 15) hypothesizes that cryptochrome uses a mechanism similar to that of photolyase by donating an electron from its anionic form (FAD^– in insect or FADH^– in plant) to a putative substrate that induces a local electrostatic variation to cause conformation changes for signaling. Both models require electron transfer (ET) at the active site to induce electrostatic changes for signaling. Similar to the pyrimidine dimer, the Ade moiety near the Lf ring could also be an oxidant or a reductant. Thus, it is necessary to know the role of the Ade moiety in initial photochemistry of FAD in cryptochrome to understand the mechanism of cryptochrome signaling.

Here, we use Escherichia coli photolyase as a model system to systematically study the dynamics of the excited cofactor in four different redox forms. Using site-directed mutagenesis, we replaced all neighboring potential electron donor or acceptor amino acids to leave FAD in an environment conducive to formation of one of the four redox states. Strikingly, we observed that, in all four redox states, the excited Lf proceeds to intramolecular ET reactions with the Ade moiety. With femtosecond resolution, we followed the entire cyclic ET dynamics and determined all reaction times of wild-type and mutant forms of the enzyme to reveal the molecular origin of the active state of flavin in photolyase. With the semiclassical Marcus ET theory, we further evaluated the driving force and reorganization energy of every ET step in the photoinduced redox cycle to understand the key factors that control these ET dynamics. These observations may imply a possible active state among the four redox forms in cryptochrome.

Results and Discussion

Photoreduction-Like ET from Adenine to Neutral Oxidized (Lf) and Semiquinoid (LfH^•) Lumiflavins.

As reported in the preceding paper (16), we have shown that the excited FAD in photolyase is readily quenched by the surrounding tryptophan residues, mainly W382 with a minor contribution from W384, and that the ET dynamics from W382 to FAD* occurs ultrafast in 0.8 ps. By replacing W382 and W384 to a redox inert phenylalanine (W382F/W384F) using site-directed mutagenesis, we abolished all possible ET between FAD* and the neighboring aromatic residues and observed a dominant decay of FAD* in 19 ps (an average time of a stretched exponential decay with τ = 18 ps and β = 0.92) as shown in Fig. 2A (k_FET⁻¹) with a probing wavelength at 800 nm. The observed stretched behavior reflects a heterogeneous quenching dynamics, resulting from the coupling of ET with the active-site solvation on the similar timescales (17). The dynamics in 19 ps reflects the intramolecular ET from the Ade to Lf moieties to form a charge-separated pair of Ade⁺+Lf^–. Tuning the probe wavelengths to shorter than 700 nm to search for the maximum 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 decay dynamics reflects the charge recombination process (k_BET⁻¹) 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.

Fig. 2.

Femtosecond-resolved intramolecular ET dynamics between the excited oxidized Lf and Ade moieties. (A–C) Normalized transient-absorption signals of the W382F/W384F mutant in the oxidized state probed at 800, 630, and 580 nm, respectively, with the decomposed dynamics of the reactant (Lf*) and intermediate (Ade⁺). Inset shows the derived intramolecular ET mechanism between the oxidized Lf* and Ade moieties.

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^•* in 85 ps (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^•* in flavodoxin 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 adenine (20) and of +0.02 V vs. NHE in photolyase for neutral semiquinoid LfH^• (21), with the S₁←S₀ transition of FADH^• at 650 nm (1.91 eV) we find that the ET reaction from Ade to LfH^•* has a favorable, negative free-energy change of −0.03 eV. 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.

Fig. 3.

Femtosecond-resolved intramolecular ET dynamics between the excited neutral semiquinoid Lf and Ade moieties. (A–C) Normalized transient-absorption signals of the W382F mutant in the neutral semiquinoid state probed at 800, 555, and 530 nm, respectively, with the decomposed dynamics of two groups: one represents the excited-state (LfH^•*) dynamic behavior with the amplitude proportional to the difference of absorption coefficients between LfH^•* and LfH^•; the other gives the intermediate (Ade⁺) dynamic behavior with the amplitude proportional to the difference of absorption coefficients between Ade⁺ and LfH^•. Inset shows the derived intramolecular ET mechanism between the neutral LfH^•* and Ade moieties. For the weak signal probed at 555 nm, a long component (∼20%) was removed for clarity and this component could be from the product(s) resulting from the excited state due to the short lifetime of 230 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⁺, and LfH^• 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 coefficient (ε_LfH^•* < ε_LfH^•), 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 decay behavior and similarly the signal flips due to the larger absorption coefficient of FADH^•.

Kinetically, we observed an apparent rise in 20 ps and a decay in 85 ps. Fig. 3C shows that, when the transient is probed at 530 nm, the ground-state LfH^• recovery in 85 ps dominates the signal. Thus, the observed dynamics in 20 ps reflects the back ET process and the signal manifests as apparent reverse kinetics, leading to less accumulation of the intermediate state. Here, the charge recombination in 20 ps is much faster than the charge separation in 135 ps with a driving force of −1.88 eV in the Marcus inverted region. In summary, although the neutral FAD* and FADH^•* states can draw an electron from a strong reductant and the dimer substrate can be repaired by a strong oxidant (22) by donating an electron to induce cationic dimer splitting, the ultrafast cyclic ET dynamics with the Ade moiety in the mutants reported here or with the neighboring tryptophans in the wild type (23, 24) exclude these two neutral redox states as the functional state in photolyase.

ET from Anionic Semiquinoid Lumiflavin (Lf^–) to Adenine.

In photolyase, 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 successfully 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 exclude ET from FAD^–* to the dimer substrate (SI Text).

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 difference of absorption coefficients between (Lf+Ade^–) and Lf^–. Inset shows the derived intramolecular ET mechanism between the anionic Lf^–* and Ade moieties.

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 12 ps must result from an intramolecular ET from Lf^–* to Ade to 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 picoseconds, could be from an intramolecular ET with Ade as well as the ultrafast deactivation by a butterfly bending motion through a conical intersection (15, 19) due to the large plasticity of cryptochrome (28). However, photolyase is relatively rigid, and thus the ET dynamics here shows a single exponential decay with a more defined configuration.

Similarly, we tuned the probe wavelengths to the blue side to probe the intermediate states of Lf and Ade^– and minimize the total contribution of the excited-state decay components. Around 350 nm, we detected a significant intermediate signal with a rise in 2 ps and a decay in 12 ps. The signal flips to the negative absorption due to the larger ground-state Lf^– absorption. Strikingly, at 348 nm (Fig. 4C), we observed a positive component with the excited-state dynamic behavior (ε_Lf–_* > ε_Lf–) and a flipped negative component with a rise and decay dynamic profile (ε_Lf+ε_Ade– < ε_Lf–). Clearly, the observed 2 ps dynamics reflects the back ET dynamics and the intermediate signal with a slow formation and a fast decay appears as apparent reverse kinetics again. This observation is significant and explains why we did not observe any noticeable thymine dimer repair due to the ultrafast back ET to close redox cycle and thus prevent further electron tunneling to damaged DNA to induce dimer splitting. Thus, in wild-type photolyase, the ultrafast cyclic ET dynamics determines that FAD^– cannot be the functional state even though it can donate one electron. The ultrafast back ET dynamics with the intervening Ade moiety completely eliminates further electron tunneling to the dimer substrate. Also, this observation explains why photolyase uses fully reduced FADH^– as the catalytic cofactor rather than FAD^– even though FAD^– can be readily reduced from the oxidized FAD.

ET from Anionic Hydroquinoid Lumiflavin (LfH^–) to Adenine.

Previously, we reported the total lifetime of 1.3 ns for FADH^–* (2). Because the free-energy change ΔG⁰ for ET from fully reduced LfH^–* to adenine is about +0.04 eV (5, 21), the ET dynamics could occur on a long timescale. We observed that the fluorescence 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 positive (ε_LfH^•+ε_Ade– > ε_LfH–+ε_Ade) and dominant. Similarly, we observed an apparent reverse kinetics with a rise in 25 ps and a decay in 1.3 ns. With the N378C mutant, we reported the lifetime of FADH^–* as 3.6 ns (4) and taking this value as the lifetime without ET with the Ade moiety, we obtain the forward ET time as 2 ns. Thus, the rise dynamics in 25 ps reflects the back ET and this process is ultrafast, much faster than the forward ET. This observation is significant and indicated that the ET from the cofactor to the dimer substrate in 250 ps does not follow the hopping 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).

Fig. 5.

Femtosecond-resolved intramolecular ET dynamics between the excited anionic hydroquinoid Lf and Ade moieties. (A–C) Normalized transient-absorption signals in the anionic hydroquinoid state probed at 800, 270, and 269 nm with the decomposed dynamics of two groups: one represents the excited-state (LfH^–*) dynamic behavior with the amplitude proportional to the difference of absorption coefficients between LfH^–* and LfH^–; the other reflects the intermediate (LfH^• or Ade^–) dynamic behavior with the amplitude proportional to the difference of absorption coefficients between (LfH^•+Ade^–) and (LfH^–+Ade). Inset shows the derived intramolecular ET mechanism between the anionic LfH^–* and Ade moieties.

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 plant cryptochrome, then the intramolecular ET dynamics with the Ade moiety could be significant due to the charge relocation to cause an electrostatic change, even though the back ET could be ultrafast, and such a sudden variation could induce local conformation changes to form the initial signaling state. Conversely, if the active state is FAD, the ET dynamics in the wild type of cryptochrome is ultrafast at about 1 ps with the neighboring tryptophan(s) and the charge recombination is in tens of picoseconds (15). Such ultrafast change in electrostatics could be similar to the variation induced by the intramolecular ET of FAD^–* or FADH^–*. Thus, the unusual bent configuration assures an “intrinsic” intramolecular ET within the cofactor to induce a large electrostatic variation for local conformation changes in cryptochrome, which may imply its functional role.

We believe the findings reported here explain why the active state of flavin in photolyase is FADH^–. With the unusual bent configuration, the intrinsic ET dynamics determines the only choice of the active state to be FADH^–, not FAD^–, due to the much slower intramolecular ET dynamics within the cofactor in the former (2 ns) than in the latter (12 ps), although both anionic redox states could donate one electron to the dimer substrate. With the neutral redox states of FAD and FADH^•, the ET dynamics are ultrafast with the neighboring aromatic tryptophan(s) even though the dimer substrate could donate one electron to the neutral cofactor, but the ET dynamics is not favorable, being much slower than those with the tryptophans or the Ade moiety. Thus, the only active state for photolyase is anionic hydroquinone FADH^– with an unusual, bent configuration due to the unique dynamics of the slower intramolecular ET (2 ns) in the cofactor and the faster intermolecular ET (250 ps) with the dimer substrate (4). These intrinsic intramolecular cyclic ET dynamics in the four redox states are summarized in Fig. 6A.

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).

Energetics of ET in Photolyase Analyzed by Marcus Theory.

The intrinsic intramolecular ET dynamics in the unusual bent cofactor configuration with four different redox states all follow a single exponential decay with a slightly stretched behavior (β = 0.90–0.97) due to the compact juxtaposition of the flavin and Ade moieties in FAD. Thus, these ET dynamics are weakly coupled with local protein relaxations. With the cyclic forward and back ET rates, we can use the semiempirical Marcus ET theory (30) as treated in the preceding paper (16) and evaluate the driving forces (ΔG⁰) and reorganization energies (λ) for the ET reactions of the four redox states. Because no significant conformation variation in the active site for different redox states is observed (31), we assume that all ET reactions have the similar electronic coupling constant of J = 12 meV as reported for the oxidized state (16). With assumption that the reorganization energy of the back ET is larger than that of the forward ET, we solved the driving force and reorganization energy of each ET step and the results are shown in Fig. 6B with a 2D contour plot. The driving forces of all forward ET fall in the region between +0.04 and −0.28 eV, whereas the corresponding back ET is in the range from −1.88 to −2.52 eV. The reorganization energy of the forward ET varies from 0.88 to 1.10 eV, whereas the back ET acquires a larger value from 1.11 to 1.64 eV. These values are consistent with our previous findings about the reorganization energy of flavin-involved ET in photolyase (5), which is mainly contributed by the distortion of the flavin cofactor during ET (close to 1 eV). All forward ET steps fall in the Marcus normal region due to their small driving forces and all of the back ET processes are in the Marcus inverted region. Note that the back ET dynamics of the anionic cofactors (2 and 4 in Fig. 6B) have noticeably larger reorganization energies than those with the neutral flavins probably because different high-frequency vibrational energy is involved in different back ETs. Overall, the ET dynamics are controlled by both free-energy change and reorganization energy as shown in Fig. 6B. The active site of photolyase modulates both factors to control the ET dynamics of charge separation and recombination or charge relocations in every redox state.

Conclusion

We reported here our direct observation of intramolecular ET between the Lf and Ade moieties with an unusual bent configuration of the flavin cofactor in photolyase in four different redox states using femtosecond spectroscopy and site-direct mutagenesis. Upon blue-light excitation, the neutral oxidized and semiquinone lumiflavins can be photoreduced by accepting an electron from the Ade moiety (or neighboring aromatic tryptophans), while the anionic semiquinone and hydroquinone lumiflavins can reduce the Ade moiety by donating an electron. After the initial charge separation or relocation, all back ET dynamics occur ultrafast in less than 100 ps to close the photoinduced redox cycle. Strikingly, in contrast to the oxidized state, all other three back ET dynamics are much faster than their forward ET processes, leading to less accumulation of the intermediate state. To capture the intermediate states, it is necessary to find an appropriate probing wavelength to cancel out the contributions from both the excited state (positive signal) and ground state (negative signal), leaving the weak intermediate signal dominant.

The intramolecular ET dynamics in the four redox states with the bent cofactor configuration reveal the molecular origin of the active state in photolyase and imply a universal ET model for both photolyase and cryptochrome. To repair damaged DNA in photolyase, the ET must be from the anionic flavin cofactor and the intramolecular ET dynamics unambiguously reveal that only the FADH^– as the active state rather than FAD^– due to the intrinsically slower ET (2 ns) in the former and faster ET (12 ps) in the latter, allowing a feasible, relatively fast, ET (250 ps) to the damaged-DNA substrate from FADH^– with the intervening Ade moiety in the middle to mediate such initial electron tunneling for repair. In cryptochrome, either neutral FAD and FADH^• or anionic FAD^– and FADH^– can proceed to an ET dynamics upon blue-light excitation. For the former, the ET with the neighboring aromatic tryptophans occur in 1 and 45 ps or with the Ade moiety in 19 and 135 ps, and for the latter, the ET with the Ade moiety occur in 12 ps and 2 ns, respectively. All back ET dynamics occur within 100 ps. Such ET dynamics induce an electrostatic variation in the active site, leading to local conformation changes to form the initial signaling state. A unified ET mechanism for both photolyase and cryptochrome would imply that an anionic redox form is more attractive as a functional state in cryptochrome. Further studies are needed, however, to understand the signaling mechanism(s) of photosensory cryptochrome.

Materials and Methods

Photolyase Mutants and Their Redox States.

The preparation of His-tag fused E. coli photolyase E109A mutant (EcPL) has been described before (32). Based 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 anionic semiquinone (FAD^–), we mutated two critical positions in EcPL of E363L and N378C. After purification, we obtained the mutant protein in completely oxidized state. Before ultrafast experiments, the mutant enzyme of a concentration of 100 μM was exchanged into a basic reaction buffer at pH 9 with 50 mM Tris⋅HCl, 300 mM NaCl, 1 mM EDTA, 20 mM DTT, 1 mM oligo-(dT)₁₅ containing cyclobutane thymine dimers, and 50% (vol/vol) glycerol. After purge with argon and irradiation with UV light at 365 nm (UVP; 8 W), the flavin cofactor is stabilized at the FAD^– state under anaerobic conditions. The neutral semiquinone (FADH^•) EcPL was prepared by mutation of W382F in EcPL and the anionic hydroquinone (FADH^–) EcPL was stabilized under anaerobic conditions after purge with argon and subsequent photoreduction.

Femtosecond Absorption Spectroscopy.

All of the femtosecond-resolved measurements were carried out using the transient-absorption method. The experimental layout has been detailed previously (24). Enzyme preparations with oxidized (FAD) and anionic semiquinone (FAD^–) flavin were excited at 480 nm. For enzyme with neutral semiquinone (FADH^•), the pump wavelength was set at 640 nm. For the anionic hydroquinone (FADH^–) form of the enzyme, we used 400 nm as the excitation wavelength. The probe wavelengths were tuned to cover a wide range of wavelengths from 800 to 260 nm. The instrument time resolution is about 250 fs and all of the experiments were done at the magic angle (54.7°). Samples were kept stirring during irradiation to avoid heating and photobleaching. Experiments with the neutral FAD and FADH^• states were carried out under aerobic conditions, whereas those with the anionic FAD^– and FADH^– states were executed under anaerobic conditions. All experiments were performed in quartz cuvettes with a 5-mm optical length except that the FADH^– experiments probed at 270 and 269 nm were carried out in quartz cuvettes with a 1-mm optical length.