Dynamics and mechanism of repair of ultraviolet-induced (6–4) photoproduct by photolyase

Abstract

The enzyme (6-4) photolyase utilizes blue-light energy to repair DNA damage by cleaving the ultraviolet-induced bond between pyrimidine dimers. Ultrafast spectroscopy has now been used to examine the detailed electron and proton movements during the repair photocycle. Histidine 364 is identified as the crucial residue involved in the rate-limiting step. The repair enzyme (6–4) photolyase uses light energy to cleave the ultraviolet-induced bond between pyrimidine dimers. These authors use ultrafast spectroscopy to examine the detailed electron and proton movements during the catalytic photocycle. Histidine 364 is identified as the crucial residue involved in the rate-limiting step. One of the detrimental effects of ultraviolet radiation on DNA is the formation of the (6–4) photoproduct, 6–4PP, between two adjacent pyrimidine rings1. This lesion interferes with replication and transcription, and may result in mutation and cell death2. In many organisms, a flavoenzyme called photolyase uses blue light energy to repair the 6–4PP (ref. 3). The molecular mechanism of the repair reaction is poorly understood. Here, we use ultrafast spectroscopy to show that the key step in the repair photocycle is a cyclic proton transfer between the enzyme and the substrate. 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. These key dynamics define the repair photocycle and explain the underlying molecular mechanism of the enzyme’s modest efficiency.