Electron Nuclear Double Resonance Differentiates Complementary Roles for Active Site Histidines in (6-4) Photolyase^*

Abstract

(6-4) photolyase catalyzes the light-dependent repair of UV-damaged DNA containing (6-4) photoproducts. Blue light excitation of the enzyme generates the neutral FAD radical, FADH·, which is believed to be transiently formed during the enzymatic DNA repair. Here (6-4) photolyase has been examined by optical spectroscopy, electron paramagnetic resonance, and pulsed electron nuclear double resonance spectroscopy. Characterization of selected proton hyperfine couplings of FADH·, namely those of H^8α and H^1′, yields information on the micropolarity at the site where the DNA substrate is expected to bind. Shifts in the hyperfine couplings as a function of structural modifications induced by point mutations and pH changes distinguish the protonation states of two highly conserved histidines, His³⁵⁴ and His³⁵⁸, in Xenopus laevis (6-4) photolyase. These are proposed to catalyze formation of the oxetane intermediate that precedes light-initiated DNA repair. The results show that at pH 9.5, where the enzymatic repair activity is highest, His³⁵⁸ is deprotonated, whereas His³⁵⁴ is protonated. Hence, the latter is likely the proton donor that initiates oxetane formation from the (6-4) photoproduct.