Flavins dimers

Although the reduction potentials of DNA bases and UV induced DNA lesions inside a DNA double strand or inside the active site of a DNA photolyase, together with the reduction potential of the photoexcited FADH- in the photolyases, are not known, currently available redox potentials indicate that the single electron reduction of a nucleobase or a UV induced dimer lesion by a reduced and deprotonated flavin coenzyme is a weakly exothermic process. The reduced and deprotonated FADH- in its photoexcited state is... 

Scheme 2 Mechanism of repair of cyclobutane pyrimidine dimers (CPD) by a CPD photolyase. 8-HDF 8-hydroxy-5-deazaflavin, ET electron transfer. FADH reduced and de-protonated flavin-coenzyme...

In order to investigate the single electron donation process from a reduced flavin to a pyrimidine dimer or oxetane lesion, the photolyase model compounds 1-4 depicted in Scheme 4 were prepared [41, 42]. The first model compounds 1 and 2 contain a cyclobutane uracil (1) or thymine (2) dimer covalently connected to a flavin, which is the active electron donating subunit in photolyases. These model systems were dissolved in various solvents... 

Scheme 4 The four pyrimidine dimer and oxetane model compounds 1-4 with either a reduced and deprotonated flavin, or a pyrene as the electron donor. Depiction of the detected reaction products 5-7...

These experiments proved that a light-excited, reduced flavin is indeed able to photoreduce cyclobutane pyrimidine dimers and that these dimers undergo a spontaneous cycloreversion. The quantum yield of about 0=5% clarified that the overall dimer splitting process is highly efficient, even in these simple model systems ((]) photolyase 70%). 

Whatever the reason may be behind the strict necessity to deprotonate the flavin donor, the reduced and deprotonated flavin was established in these model studies to be an efficient electron donor, able to reduce nucle-obases and oxetanes. In the model compounds 1 and 2 the pyrimidine dimer translates the electron transfer step into a rapidly detectable chemical cycloreversion reaction [47, 48], Incorporation of a flavin and of a cyclobutane pyrimidine dimer into DNA double strands was consequently performed in order to analyse the reductive electron transfer properties of DNA. 

Incorporation of a flavin electron donor and a thymine dimer acceptor into DNA double strands was achieved as depicted in Scheme 5 using a complex phosphoramidite/H-phosphonate/phosphoramidite DNA synthesis protocol. For the preparation of a flavin-base, which fits well into a DNA double strand structure, riboflavin was reacted with benzaldehyde-dimethylacetale to rigidify the ribityl-chain as a part of a 1,3-dioxane substructure [49]. The benzacetal-protected flavin was finally converted into the 5 -dimethoxytri-tyl-protected-3 -H-phosphonate ready for the incorporation into DNA using machine assisted DNA synthesis (Scheme 5a). For the cyclobutane pyrimidine dimer acceptor, a formacetal-linked thymine dimer phosphoramidite was prepared, which was found to be accessible in large quantities [50]. Both the flavin base and the formacetal-linked thymidine dimer, were finally incorporated into DNA strands like 7-12 (Scheme 5c). As depicted in... 

Scheme 5 a Flavin-H-phosphonate and formacetal-linked thymine dimer phospho-ramidite used for the synthesis of the flavin and dimer containing DNA-strands 7-12. b Representation of a reduced flavin- and formacetal-linked cyclobutane pyrimidine dimer containing DNA strand, which upon irradiation (hv) and electron transfer (ET) performs a cycloreversion (CR) of the dimer unit, c Depiction of the investigated oligonucleotides... 

The cycloreversion experiments showed a clean Tf=T-DNA to T/T-DNA transformation. No by-products were detected, which supports the idea that DNA may be more stable towards reduction compared to oxidation. Even heating the irradiated DNA with piperidine furnished no other DNA strands other then the repaired strands, showing that base labile sites - indicative for DNA damage - are not formed in the reductive regime. The quantum yield of the intra-DNA repair reaction was therefore calculated based on the assumption that the irradiation of the flavin-Tf=T-DNA strands induces a clean intramolecular excess electron transfer driven cycloreversion. The quantum yield was found to be around 0=0.005, which is high for a photoreaction in DNA. A first insight into how DNA is able to mediate the excess electron transfer was gained with the double strands 11 and 12 in which an additional A T base pair compared to 7 and 8 separates the dimer and the flavin unit. 

In strand 13, the flavin donor and the dimer acceptor are adjacent to each other, located at a distance of 3.4 A. In the other four strands 14-17, the dis-... 

Scheme 6 a Incorporation of a silyl-spaced dimer into DNA, and treatment of the DNA strand with fluoride furnished DNA strand containing a dimer-unit with an open backbone. b Schematic representation of the light (ho) induced excess electron transfer (ET) from the reduced flavin to the dimer, followed by cycloreversion (CR). c The five DNA duplexes 13-17 containing the flavin donor and the dimer acceptor at increasing distances, together with the measured repair yields after irradiation for 1 min... 

Fig. 4 Flavin amino acids FI and F2 used for the preparation of DNA PNA hybrid duplexes. These duplexes possess the flavin in the PNA strand and the backbone opened dimer in the DNA strand. a Indicates unstructured melting. M.p.= melting points of the constructs...

In order to gain a more detailed insight into how the flavin and thymine dimer orientation may influence the excess electron transfer process, rigid... 

Fig. 5 Flavin-capped and dimer containing DNA hairpin for excess electron transfer studies through DNA...

Flavin-cyclobutane pyrimidine dimer and flavin-oxetane model compounds like 1-3 showed for the first time that a reduced and deprotonated flavin is a strong photo-reductant even outside a protein environment, able to transfer an extra electron to cyclobutane pyrimidine dimers and oxetanes. There then spontaneously perform either a [2n+2n cycloreversion or a retro-Paternd-Buchi reaction. In this sense, the model compounds mimic the electron transfer driven DNA repair process of CPD- and (6-4)-photolyases. 

Incorporation of an artificial flavin nucleobase and of a cyclobutane pyrimidine dimer building block into DNA DNA double strands, DNArPNA hybrid duplexes, and DNA-hairpins, provided compelling evidence that an excess electron can hop through DNA to initiate dimer repair even at a remote site. The maximum excess electron transfer distance realised so far in these defined Donor-DNA-Acceptor systems is 24 A. New experiments are now in progress to clarify whether even larger transfer distances can be achieved. 

Three other proteins with similar domain structure as that of FprA were reported in other bacteria (WasserfaUen et al. 1995 Gomes et al. 1997, 2000). The recombinant CthFprA and CthHrb, overexpressed in E. coli, were purified and characterized. Both FprA and Hrb were found to be present as dimers. Metal/cofactor analysis of the purified proteins revealed the presence of 2 mol each of iron and flavin (FMN) per mole dimer of Hrb and 4 mol of iron and 2 mol FMN per mole dimer of FprA. The EPR spectra of the purified proteins indicated that iron is present in a di-iron center in FprA and as a Fe(Cys)4 cluster in Hrb. 

Fig. 13. Schematic representation of the overall NOS architecture and summary of work presented in 139). Heterodimers were generated to test if electron transfer from the FMN domain proceeds via an inter- or intrasubunit process. When holo-NOS containing an inactive heme domain was dimerized with an active heme domain, activity was observed. However, when active holo-NOS was dimerized with the inactive heme domain, no activity was observed. These results indicate that the flavin domain of monomer A transfers electrons to the heme domain of monomer B.

Knowledge of the enzymes used by microorganisms in the transformation of nitroaromatic compounds is limited. Blasco Castillo (1993) characterized an inducible nitrophenol reductase from Rhodobacter capsulatus that catalyzed the reduction of 2,4-dinitrophenol (DNP) to 2-amino-4-nitrophenol. This enzyme was a dimer that contained flavin mononucleotide and possibly nonheme iron as... 

The phenolics ( + )catechin and (— )epicatechin are common flavanols in several fruits (128). Apples and pears contain other phenolic compounds such as quinic, shikimic, chlorogenic, and caffeic acids (39). Durkee and Poapst (162) reported that the two major phenolic constituents of core tissues and seeds of McIntosh apples were chlorogenic acid and phloridzin. After hydrolysis of extracts from core tissues, the identified phenolics were phloretin, caffeic acid, p-coumaric acid, phloretic acid, and trace amounts of ferulic acid. Studies have shown that apple leucoanthocyanins yield catechin, epicatechin, cyanidin, and pelargonidin after hydrolysis (163, 164). Van Buren et al. (164) also reported that a purified leucoanthocyanin from apples was either a dimer or oligomer containing ( —) epicatechin, and 5,7,3, 4 -flavin-3,4-diol. 

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