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 UVdamaged DNA containing (6-4) photoproducts. Blue light excitation of the enzyme generates the neutral FAD radical, FADH ⅐ , 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 ⅐ , namely those of H 8␣ 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, His 354 and His 358 , 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, His 358 is deprotonated, whereas His 354 is protonated. Hence, the latter is likely the proton donor that initiates oxetane formation from the (6-4) photoproduct.Ultraviolet light ( Յ300 nm) damages cellular DNA by the formation of cyclobutane pyrimidine dimers (CPDs) 3 and (6-4) photoproducts from adjacent pyrimidine bases on the same DNA strand (1). Such dimers are restored to their monomeric form by the action of two photoactive (300 Ͻ Ͻ 500 nm) damage-specific DNA repair enzymes, named CPD photolyase and (6-4) photolyase, collectively known as DNA photolyases (2-6). Both enzymes are found in various organisms, exhibit a 20 -30% amino acid sequence identity (2,7,8), and share a common chromophore, FAD (9 -12), although the two photolyases differ in DNA substrate specificity and repair mechanism.For the CPD photolyase, the initial step in the proposed repair mechanism (13) is a photoinduced electron transfer from the fully reduced FAD cofactor (FADH Ϫ ) to the CPD, resulting in the formation of a CPD anion radical and a neutral FADH ⅐ radical. The cyclobutane ring of the unstable CPD radical opens, and subsequently the electron is transferred back to the FADH ⅐ radical, thus restoring the initial redox states of both the FAD and the pair of pyrimidine bases in the DNA. Thus the entire process represents a true catalytic cycle with net-zero exchanged electrons.Unlike CPD photolyase, (6-4) photolyases are not able to directly restore the original bases from the (6-4) photoproduct in UV-damaged DNA; rather, following binding of the lesion, the overall repair reaction consists of two distinct steps, one of which is light-independent and the other one light-dependent ( Fig. 1) (14 -16). In the initial light-independent step, a 6Ј-iminium ion is thought to be generated via proton transfer induced by two histidines highly conserved among the (6-4) photolyases (His 354 and His 358 in Xenopus laevis (6-4) photolyase) (16) (Fig. 2). This intermediate spontaneously rearranges to form an ox...

Functional motifs in the (6-4) photolyase crystal structure make a comparative framework for DNA repair photolyases and clock cryptochromes

“…The second (At64PHR His-364) and final (His-368) His residues of this motif (Fig. 4B) are critical for DNA repair (9,12), as shown by the loss of activity upon mutation (Fig. S3).…”

“…CPD and (6-4) PHRs share many functional similarities, but have evolved distinct substrate specificities and mechanisms (9)(10)(11)(12). CPD and (6-4) photoproducts (Fig.…”

Proton Transfer to Flavin Stabilizes the Signaling State of the Blue Light Receptor Plant Cryptochrome

“…This proton transfer is necessary for the photoreduction of photolyases from the oxidized to the fully reduced state as well without stabilization of the neutral radical state (57,58). Most likely, proton transfer is mediated by bulk water and transferred via structural water and hydrogen bonding pathways to flavin upon photoreduction.…”

Studies of Organic Protein Cofactors Using Electron Paramagnetic Resonance

“…16. 128 The hyperfine splittings of the H(8) and H(1 0 ) protons shifted significantly as a function of structural modifications induced by point mutations and pH changes. In the absence of an X-ray structure for a (6-4) photolyase, we proposed that His354 is close to H(1 0 ) of the FADH , and His358 is close to the H(8) methyl protons.…”

“…That the H(8) proton hyperfine coupling of the FADH radical in (6-4) photolyase is even smaller than that of E. coli DNA photolyase, 128 implies an even less polar and/or more aromatic environment of this enzyme, which might be due to one or both of the following changes relative to the E. coli enzyme: (i) a smaller substrate-binding pocket for (6-4) photoproducts in (6-4) photolyase that would limit the number of water molecules that could approach the redox-active flavin cofactor in the absence of a DNA lesion; and (ii) two aromatic histidine residues, His354 and His358, that are predicted to replace non-aromatic Asn and Met in E. coli photolyase render the cofactor binding site more aromatic. These histidines play a role in catalyzing the formation of an oxetane intermediate from the photoproduct that precedes light-initiated DNA repair, see Fig.…”