Probing Electrostatics in a DNA Repair Enzyme with a pH-Sensitive Nitroxyl Spin Label

Enzymes use electrostatic interactions
to recognize their substrates,
preorganize active sites, and stabilize reaction transition states.
Formamidopyrimidine–DNA glycosylase (Fpg) is a bacterial enzyme
that repairs a pro-mutagenic DNA lesion, 8-oxoguanine; its human homologues
are important for cancer prevention. General acid–base catalysis
in the active site of Fpg requires a finely tuned proton transfer
between Pro1 and Glu2 residues. To assess the protonation state of
the active site experimentally, we used EPR spectroscopy with a novel
imidazolidine nitroxyl spin label responsive in the physiological
pH range. The label showed excellent sensitivity in detecting DNA
duplex formation and Fpg binding, allowing us to measure changes in
the local electrostatic potential. We constructed DNA duplexes placing
the spin label near the active site, as verified by EPR and molecular
dynamics. Comparing the pH response of the label bound to wild-type
Fpg and its catalytically dead E2Q mutant, we observed a notable deviation
at pH 7.00 and above, which provides experimental evidence for a mutation-induced
shift in the local electrostatic potential at the spin label site.
Thus, our approach allows indirect but sensitive probing of the ionization
state of key catalytic residues. This work demonstrates that pH-sensitive
spin labels hold great potential for exploring electrostatic interactions
in DNA–protein complexes, where fine-tuning of local charge
is critical for function.