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1. Abstract
This paper aims to describe the mechanism and unique catalytic process of Photolyase enzymes. This unique class of enzymes main function in organism is to act on DNA, largely as a repair system. While this is hardly unique, photolyases achieve this the enzyme uses photons on the blue side of the visible spectrum and UV radiation to activate and power their reactions.

DNA absorbs photons at approximately~265 nm, the UV-C range, with a tail extending beyond 290. This causes UV lesions which inhibit DNA replication and transcription, thus leading to mutagenesis, carcinogenesis and cell death. while there are many varieties of base damage, among the most common are the photocycloaddition reactions between two adjacent pyrimidines, which gives rise to the formation of cyclobutane pyrimidine dimers and pyrimidine(6–4)pyrimidone photoproducts, also called (6–4) photoproducts. Photolyases repair these lesions, among others, with high specificity using blue light photons.

2. DNA Lesion Repair
In 1949, a phenomenon named photoreactivation whereby bacteria inactivated by UV irradiation could be reactivated when exposed to visible light It was soon demonstrated that photoreactivation is caused by an enzyme. It was later named DNA photolyase (PL) and was found in organisms from all kingdoms of life, with the remarkable exception of placental mammals. The enzyme acts by electron transfer using a reduced flavin FADH− which is activated by light energy to act as an electron donor to break pyrimidine dimer lesions.

4 Structure
Photolyase is a flavoprotein, found bound to a coenzyme, typically FADH. In E. coli for example, although these enzymes are found in a vast range of organisms, the enzyme consists of a single polypeptide chain of 471 amino acids with two well-defined domains:  an N-terminal α/β domain (residues 1−131) and a C-terminal α-helical domain (residues 204−471), which are connected to one another with a long interdomain loop (residues 132−203) that wraps around the α/β domain. Often noncovalently bonded prosthetic groups are attached to the enzyme. In E. coli one is a light-harvesting photoantenna, a pterin molecule in the form of methenyltetrahydrofolate (MTHF), and the other one is the catalytic cofactor, a fully reduced deprotonated flavin molecule FADH-. The MTHF photoantenna is located between the two domains and partially sticks out from the surface of the enzyme. THis is an example of a folate photolyase, although there is another class, deazaflavin photolyases, which contain 8-hydroxy-7,8-didemethyl-5-deazariboflavin (8-HDF) instead of MTHF. Only FAD is required for catalytic activity, however the second cofactor significantly accelerates reaction rate in low-light conditions. The FADH is also often photoactive in many enzymes, and participates in the mechanism as a photon acceptor. Despite a variety of evolutionarily distinct photolyases, proteins show strong sequence homology

5. Mechanism
While the mechanism of photolyases always require a FADH cofactor, and a conformational "flip out" of damaged bases into the active site of the enzyme, there are necessarily differences in exact mechanism for different classes of photolyase repair. While some, like CPD photolyase have found general consensus, others like, (6-4) photolyase, are more controversial and are still being examined. These photolyases can be used to describe the typical mechanism of action of this class of enzymes, as well as to paint a picture of how the complexity of their function makes studying their mechanisms challenging.

5.1 Cyclobutane Pyrimidine Dimer (CPD) Photolyase
Photolyases bind CPD-containing DNA substrates in a cleft with dissociation constant (Kd) of the order of 10^-9 M and an association rate constant (kon) of the order of 10^7 M^-1s^-1. Photolyase flips the CPD lesion out of the DNA duplex and aligns in the active site proximal to FAD for effective electron transfer. Once bound, reduced FADH is excited, and an electron is transferred to the CPD in ~30– 250 ps, forming an anion radical of the CPD and a semireduced FADH radical. The cyclobutane ring then splits at a high rate, rearranging into an intact base and the anion radical of the other base, as studied in Liu et. al 5' and 3' respectively. The radical is returned to FADH in ~1 ns, repairing the DNA and restoring FADH. CPD repair by photolyase is a single-photon process with a high (40–100%) quantum yield.

5.2 (6-4) photolyase
(6-4) lesion repair is a more challenging story, potentially involving a second photoexcitation of the photolyase. The repair photocycle is a cyclic proton transfer between the enzyme and the substrate, and FADH- remains the key catalytic cofactor Similarly to CPD photolyase, (6-4) PP-containing substrates are bound in a conserved manner, with Kd of the order of 10^-9 M. Repaired DNA was determined to be released at a similar rate to the CPD case however, the quantum yield of photorepair of (6-4) PPs was found to be ~4 times or even ~100 times lower than for CPD repair.

Based on the homology between photolyases, it was initially proposed that (6-4) photolyases first catalyze the formation of a four membered ring, similar to the cyclobutane for CPDs, in the dark, the same oxetane intermediate as found in CPD. Photorepair would then follow just as for CPD by electron transfer from the excited FADH to the transformed lesion. However, later structural studies showed that an oxetane was not formed, and conserved histidine residues (H365 and H369 in Dm64) thought to be responsible for stabilizing the oxetane instead were seen to form, together with a conserved tyrosine residue (Y423 in Dm64), a hydrogen bond network, proximal to the lesion, stabilizing its position. To resolve this, new mechanisms were proposed, including a “transient water pathway” where upon formation of the radical of the (6–4) PP, the O4' located at towards the 5' wouldprotonate via His1, and a water molecule would attack C4' at the 3' component, thereby transferring the O4'H functional group. Splitting of the C6-C4 bond would follow, accompanied by the return of excess electron (to FADH) and loss of a proton.

Computational studies did not readily confirm this “transient water pathway” because of a computed high activation barrier, however variants of this mechanism are still under consideration. Domratcheva et al. proposed a variant of the transient water pathway based on a direct transfer of the hydroxyl group (O4'H), from the 5' to the 3'. The mechanism was later expanded, including the His1 residue (H365 in Dm64) in the calculations Faraji et al. proposed another (series of) modification to the transient water pathway where OH transfer is preceded by the protonation of the N3' atom of the 3' base by the protonated His1.Essentially the proton transfer was "steered". Another computational route was proposed by Sadeghian et al. where first photoexcitation yields the oxetane intermediate, which is repaired by the second photoexcitation. Sadeghian et al. claimed that (6-4) PPs could not be repaired by photolyase upon one single photoexcitation. The first reaction steps are very similar to the “proton-transfer-steered OH transfer”, however, instead of a direct transfer of the O4' H group, O4' then forms a bond with C40 while keeping a bond with C5, yielding the oxetane-bridged dimer in its ground state. Re-excitation of FADH by a second photon will transfer an electron into the oxetane intermediate, forming a radical anion. The C5-O4' bond then splits with protonation of O4' by His1H+, allowing the O4' H group to transfer to the 3' base.

With no evident computational winner, the study of this enzymes's mechanism was forced to resort to experiment. Luckily, the light absorbing properties necessary for excitation make this system an ideal candidate for spectroscopy. (this is good because lasers are cool). Li et al. measured primary reactions related to the repair of a T(6-4)T lesion of the (6-4) photolyase of Arabidopsis thaliana (At64), using ultrafast fluorescence and transient absorption spectroscopy between 315 and 800 nm. They observed electron transfer to the (6-4) PP following excitation of FADH in 225 ps, interestingly, ~90% of the FADH radicals formed by this primary electron transport event, in stark contrast to the CPD mechanism, were re-reduced very quickly (in 50 ps). The remainder of the FADH did not decay in the accessed time window for a long duration (up to 3 ns). Transient absorption changes in the 315-370 nm range were used to assign a proton transfer from (protonated) His1 (His364 in At64) to the reduced substrate, occurring in 425 ps.

As to whether these kinetic measurements can be used to tell whether this is a one or two excitation process, it is hard to say. It is not easy to distinguish between the "ordinary" one-photon mechanism and a sequential two-photon. Li et. al used non-saturating 100-ps laser flashes where the pulse duration was shorter than the reported lifetime of the excited flavin in the presence of substrate (225 ps (68)), which theoretically excludes double excitations of FADH within one flash to try to provide evidence of double excitation mechanism. The effect of their first flash was to depleted the T(6-4)T lesion without yielding substantial repair while subsequent flashes, sent after a pre-irradiation burst, simultaneously depleted the T(6-4)T lesion and produced repair. Li et al. observe quadratic behavior at 265 nm with of series of 20 flashes suggesting that photorepair by (6-4) photolyase requires two photoexcitations.

9. Spectroscopy and kinetics
By femtosecond synchronization of the enzymatic dynamics with the repair function, we followed the function evolution and observed direct electron transfer from the excited flavin cofactor to the 6–4PP in 225 picoseconds, but surprisingly fast back electron transfer in 50 picoseconds without repair. We found that the catalytic proton transfer between a histidine residue in the active site and the 6–4PP, induced by the initial photoinduced electron transfer from the excited flavin cofactor to 6–4PP, occurs in 425 picoseconds and leads to 6–4PP repair in tens of nanoseconds. Category:Pigments Category:Color Category:Photochemistry