Computational Assignment of the Histidine Protonation State in (6-4) Photolyase Enzyme and Its Effect on the Protonation Step

The initial step to resolving the controversy regarding the repair mechanism of the pyrimidine (6-4) pyrimidone photoproduct DNA lesion is to determine the protonation state of the two conserved active site histidine residues (His365 and His369 in Drosophila melanogaster) in the (6-4) photolyase enzyme ((6-4) PHR). Both His residues were experimentally determined to be crucial for catalysis. Most previous theoretical studies assumed the presence of protonated His365 in the active site, which would transfer its proton to N3′ of the lesion as the first step in repair; however, other empirical/semiempirical pK a calculations suggested the presence of two neutral His residues in the active site. Here, we conduct molecular dynamics simulations (MD) of the (6-4) PHR/DNA complex on the basis of the X-ray crystal structure to investigate all combinations of the three possible protonation states of each His. The MD results show that HIP365 (both ND1 and NE2 protonated) and HID369 (only ND1 protonated) are the most probable protonation states in the active site. Furthermore, we employ quantum mechanics-cluster and quantum mechanical/molecular mechanical (QM/MM) approaches to investigate the protonation of N3′, starting with three plausible complexes resulting from MD (HIP365/HID369, HID365/HIE369, and HID365/HIP369). Surprisingly, protonation of the N3′ atom is found to be feasible starting from all three protonation states: protonated HIP365 transfers the proton via a barrierless step, and in the other cases of neutral HID365, the proton is transferred from O4′ of the lesion via His365 through an energy barrier of ∼80 kJ mol–1 in both complexes. These results might explain the conservation of repair activity over a wide range of pH values.