Combined QM/MM Investigation on the Light-Driven Electron-Induced Repair of the (6–4) Thymine Dimer Catalyzed by DNA Photolyase

Abstract

The (6-4) photolyases are blue-light-activated enzymes that selectively bind to DNA and initiate splitting of mutagenic thymine (6-4) thymine photoproducts (T(6-4)T-PP) via photoinduced electron transfer from flavin adenine dinucleotide anion (FADH(-)) to the lesion triggering repair. In the present work, the repair mechanism after the initial electron transfer and the effect of the protein/DNA environment are investigated theoretically by means of hybrid quantum mechanical/molecular mechanical (QM/MM) simulations using X-ray structure of the enzyme-DNA complex. By comparison of three previously proposed repair mechanisms, we found that the lowest activation free energy is required for the pathway in which the key step governing the repair photocycle is electron transfer coupled with the proton transfer from the protonated histidine, His365, to the N3' nitrogen of the pyrimidone thymine. The transfer simultaneously occurs with concerted intramolecular OH transfer without formation of an oxetane or isolated water molecule intermediate. In contrast to previously suggested mechanisms, this newly identified pathway requires neither a subsequent two-photon process nor electronic excitation of the photolesion.

Characterization of the Intermediate in and Identification of the Repair Mechanism of (6‐4) Photolesions by Photolyases

“…Here, 6‐4PP is assumed to be converted to an oxetane ring, which requires the attack of the O4′ oxygen at the C4′ carbon involving intermediate I (Figure ). II) The transient water mechanism, in which the nearby His365 residue plays a crucial role, since proton transfer from this protonated His365 to O4′ is postulated to precede the cleavage of H 2 O4′ from the 5′ thymine involving intermediate IIb (Figure ). III) The so‐called proton transfer steered mechanism, in which proton transfer from the protonated His365 to the N3′ nitrogen of the 3′ thymine occurs simultaneously with an oxetane‐like transition state involving intermediates IIIa and IIIb (Figure ).…”

“…(Figure ). Experimental and theoretical studies have been performed intensively over the past years, to trace all steps of the repair. While the functional mechanism of CPD photolyases has been identified, the details of the repair mechanism operating in (6‐4) photolyases is still a matter of ongoing debate.…”

Characterization of the Intermediate in and Identification of the Repair Mechanism of (6‐4) Photolesions by Photolyases

“…(Figure ). Experimental and theoretical studies have been performed intensively over the past years, to trace all steps of the repair. While the functional mechanism of CPD photolyases has been identified, the details of the repair mechanism operating in (6‐4) photolyases is still a matter of ongoing debate.…”

“…Here, 6‐4PP is assumed to be converted to an oxetane ring, which requires the attack of the O4′ oxygen at the C4′ carbon involving intermediate I (Figure ). II) The transient water mechanism, in which the nearby His365 residue plays a crucial role, since proton transfer from this protonated His365 to O4′ is postulated to precede the cleavage of H 2 O4′ from the 5′ thymine involving intermediate IIb (Figure ). III) The so‐called proton transfer steered mechanism, in which proton transfer from the protonated His365 to the N3′ nitrogen of the 3′ thymine occurs simultaneously with an oxetane‐like transition state involving intermediates IIIa and IIIb (Figure ).…”

“…II) The transient water mechanism, in which the nearby His365 residue plays a crucial role, since proton transfer from this protonated His365 to O4′ is postulated to precede the cleavage of H 2 O4′ from the 5′ thymine involving intermediate IIb (Figure ). III) The so‐called proton transfer steered mechanism, in which proton transfer from the protonated His365 to the N3′ nitrogen of the 3′ thymine occurs simultaneously with an oxetane‐like transition state involving intermediates IIIa and IIIb (Figure ). IV) Neutral histidine mechanism in which OH transfer from 5′ thymine to 3′ thymine is assisted by a neutral His365 residue (see the Supporting Information (SI)).…”