Chromophore function and interaction in Escherichia coli DNA photolyase: reconstitution of the apoenzyme with pterin and/or flavin derivatives

Abstract

Native DNA photolyase, as isolated from Escherichia coli, contains a neutral flavin radical (FADH.) plus a pterin chromophore (5,10-methenyltetrahydropteroylpolyglutamate) and can be converted to its physiologically significant form by reduction of FADH. to fully reduced flavin (FADH2) with dithionite or by photoreduction. Either FADH2 or the pterin chromophore in dithionite-reduced native enzyme can function as a sensitizer in catalysis. Various enzyme forms (EFADox, EFADH., EFADH2, EPteFADox, EPteFADH., EPteFADH2, EPte) containing stoichiometric amounts of FAD in either of its three oxidation states and/or 5,10-methenyltetrahydrofolate (Pte) have been prepared in reconstitution experiments. Studies with EFADox and EPte showed that these preparations retained the ability to bind the missing chromophore. The results suggest that there could be considerable flexibility in the biological assembly of holoenzyme since the order of binding of the enzyme's chromophores is apparently unimportant, the binding of FAD is unaffected by its redox state, and enzyme preparations containing only one chromophore are reasonably stable. The same catalytic properties are observed with dithionite-reduced native enzyme or EFADH2. These preparations do not exhibit a lag in catalytic assays whereas lags are observed with preparations containing FADox or FADH. in the presence or absence of pterin. Photochemical studies show that these lags can be attributed to enzyme activation under assay conditions in a reaction involving photoreduction of enzyme-bound FADox or FADH. to FADH2. EPte is catalytically inactive, but catalytic activity is restored upon reconstitution of EPte with FADox. The results show that pterin is not required for dimer repair when FADH2 acts as the sensitizer but that FADH2 is required when dimer repair is initiated by excitation of the pterin chromophore. The relative intensity of pterin fluorescence in EPte, EPteFADH., EPteFADox, or EPteFADH2 has been used to estimate the efficiency of pterin singlet quenching by FADH. (93%), FADox (90%), or FADH2 (58%). Energy transfer from the excited pterin to flavin is energetically feasible and may account for the observed quenching of pterin fluorescence and also explain why photoreduction of FADox or FADH. is accelerated by the pterin chromophore. An irreversible photobleaching of the pterin chromophore is accelerated by FADH2 in a reaction that is accompanied by a transient oxidation of FADH2 to FADH.. Both pterin bleaching and FADH2 oxidation are inhibited by substrate.

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

“…The concentration of the resultant blue flavin radical (FADH ⅐ ) was estimated based on its absorbance at 580 nm (⑀ 580 ϭ 0.48 ϫ 10 4 M Ϫ1 cm Ϫ1 ) (27). Buffer Exchange-Samples were transferred into the desired buffer (usually 0.1 M NaCl, 0.05 M Tris⅐HCl, or 0.05 M HEPES (pH 6 -9.5) in 50% (v/v) glycerol) by dilution and ultrafiltration through Amicon C30 microconcentrators at 4°C.…”

Critical Role of 7,8-Didemethyl-8-hydroxy-5-deazariboflavin for Photoreactivation in Chlamydomonas reinhardtii

“…Understanding why photoreactivation in Chlamydomonas relies so heavily on FO is of interest. Previous work has demonstrated that photolyase is able to bind FAD even in the absence of a second chromophore (5,39). It appears that this is also the case for PHR2p given the following two results.…”

“…The excited FADH Ϫ reversibly transfers an electron to the damaged DNA allowing the DNA to return to its original undamaged form. Although the antenna chromophore increases the repair rate of DNA photolyase 10 -100-fold under limited-light conditions, it is generally considered nonessential for photoreactivation under standard light conditions (4, 11, 12) because DNA photolyase bound to FADH Ϫ alone is sufficient to catalyze the repair of UV-induced DNA damage (5,(13)(14)(15).FO is generated from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, an intermediate in riboflavin biosynthesis, and 4-hydroxyphenylpyruvate, a precursor to tyrosine. The reaction is catalyzed by an enzyme known as FO synthase (EC 2.5.1.77), a member of the radical S-adenosylmethionine (AdoMet) superfamily of proteins (16).…”

“…The first, FAD, is common to all photolyases. It is the catalytic chromophore, and as such, it is essential for photoreactivation (5). The identity of the second chromophore is variable, but 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO) serves this role for a number of photolyases (6 -10).…”

“…The excited FADH Ϫ reversibly transfers an electron to the damaged DNA allowing the DNA to return to its original undamaged form. Although the antenna chromophore increases the repair rate of DNA photolyase 10 -100-fold under limited-light conditions, it is generally considered nonessential for photoreactivation under standard light conditions (4,11,12) because DNA photolyase bound to FADH Ϫ alone is sufficient to catalyze the repair of UV-induced DNA damage (5,(13)(14)(15).…”