engleski

The Protonation States of the Active-Site Histidines in (6-4) Photolyase

The active sites of the (6-4) photolyases contain two conserved histidine residues, which, in the Drosophila melanogaster enzyme, correspond to His365 and His369. While there are nine combinations in which the three possible protonation states of the two his-tidines (with protons on Nd (HID), Ne (HIE), or both Nd and Ne (HIP)) can be paired, there is presently no consensus as to which of these states is present, let alone mechanistically relevant. EPR hyperfine couplings for selected protons of FADH• radical have previously been used to address this issue. Our QM/MM calculations show, however, that the experimental couplings are equally well reproduced by each of the nine combinations. Since the EPR results seemingly cannot be used to unequivocally assign the pro-tonation states, the pKa values of the two histidines were calculated using the popular PROPKA, H++ and APBS approaches, in various environments and for several lesions. These techniques consistently indicate that, at pH=7, both His365 and His369 should be neutral although the His369 is found to be more prone to become protonated. In a comparative approach, a series of molecular dynamics simulations were performed with all nine combinations, employing various reference crystal structures and different oxi-dation states of the FAD cofactor. The overall result of this approach is in agreement with our pKa results. Consequently, although the introduction of the reduced cofactor results in an increased stability for selected protonated states, particularly the His365=HID and His369=HIP combination, the neutral combination His365=HID and His365=HIE stands out as the most relevant state for the activity of the enzyme.

photolyase; pKa; EPR; MD

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