Spectroscopic and Thermodynamic Comparisons of Escherichia coli DNA Photolyase and Vibrio cholerae Cryptochrome 1

Abstract

Escherichia coli DNA photolyase and cryptochrome 1 isolated from Vibrio cholerae, a member of the CRY-DASH family, are directly compared using a variety of experimental methods including UV-vis and Raman spectroscopy, reduction potential measurements, and isothermal titration calorimetry. The semiquinone form of the cryptochrome has an absorption spectrum that is red-shifted from that of the photolyase, but the Raman spectrum indicates that the FAD binding pocket is similar to that of photolyase. The FADH(-)/FADH* reduction potential of the cryptochrome is significantly higher than that of the photolyase at 164 mV vs NHE, but it also increases upon substrate binding (to 195 mV vs NHE), an increase similar to what is observed in photolyase. The FADH(-)/FADH* reduction potential for both proteins was found to be insensitive to ATP binding. Isothermal titration calorimetry found that photolyase binds tighter to substrate (K(A) approximately 10(5) M(-1) for photolyase and approximately 10(4) M(-1) for cryptochrome 1), and the binding constants for both proteins were slightly sensitive to oxidation state. Based upon this work, it appears that this cryptochrome has significant spectroscopic and electrochemical similarities to CPD photolyase. The thermodynamic cycle of the enzymatic repair in the context of this work is discussed.

Impact of the N5-proximal Asn on the Thermodynamic and Kinetic Stability of the Semiquinone Radical in Photolyase

“…We have reported extremely sluggish kinetics for this reaction and have shown, through kinetic isotope effects, that oxidation is rate-limited by PT (33). Recent electrochemical studies, aimed at measuring the oneelectron redox potentials of CPD-PL, reported values for E 1 ranging from Ϫ48 to 0 mV, the same within experimental error for E. coli and A. nidulans CPD-PL (34,35). In these experiments, the hq-sq redox couple was titrated, with quantitative production of the sq, whereas the sq-ox couple proved inaccessible at potentials as high as ϳ400 mV.…”

“…With ⌬E ϭ E 2 Ϫ E 1 ϭ Ϫ79 mV, the maximum yield of sq under thermodynamic equilibrium would be Ͻ10%. The buildup of sq during non-equilibrium oxidation of the hq by O 2 (33) and in titrations of the hq-sq couple only (33)(34)(35) is likely a kinetic phenomenon and may be responsible for the significantly more positive E 1 values previously reported. The very negative E 2 is consistent with predictions made based on the relative binding affinities of CPD-PL for FAD sq and ox (Ϫ191 Ͼ E 2 Ͼ Ϫ242) (33).…”

“…However, little is known regarding redox regulation in these proteins. Although electrochemical potentials have been evaluated for Arabidopsis thaliana CRY1 (34) and Vibrio cholerae CRY-DASH (35), no measurements of E 2 in any PL have been reported, and the two values of E 1 currently in the literature differ by ϳ50 mV (34,35). Furthermore, it is likely that the mechanism(s) for redox regulation in PLs and CRYs involve more than adjusting the thermodynamics of ET at the FAD cofactor.…”

Fungal cryptochrome with DNA repair activity reveals an early stage in cryptochrome evolution

“…It is possible that these repair activities were the result of an experimental artifact due to the molar excess of enzyme to dsDNA and to the possibility of loop formation near the T<>T. The dsDNA probe that we used in our assays did not allow formation of secondary structures, and our controls with Arabidopsis Cry3 showed that our probes could not be repaired by a standard cry-DASH. The cry-DASH from V. cholerae (VcCry1) did not show dsDNA repair activity (19), but Sokolowsky et al (21) showed that VcCry1 could dissociate an oligo(dT) 10 /oligo(dA) 10 duplex, and monitored the increase in 260 nm absorbance as an indication of T<>T repair in the oligo(dT) 10 strand. However, an increase in 260 nm absorbance cannot distinguish between DNA strand dissociation and T<>T repair.…”

“…Cry-DASH were shown to bind and repair CPDs in single-stranded DNA or loop-structured duplex DNA (19,20), but repair of CPDs in duplex DNA by cry-DASH has only been suggested from in vitro studies (e.g., ref. 21). Cry-DASH may therefore represent an evolutionary intermediate between cryptochromes and photolyases (3,22).…”