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 H8α and H1′, 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. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.