Flavin, photoreaction

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. 

Consequently, photoreduction of dFloX leads to adduct formation according to Reaction A even in cases where normal flavins would not yield stable adducts. These adducts frequently undergo secondary photoreactions with starting dFl, as shown in B, yielding radical dimers which might obscure the picture at first sight. 

The photoreaction of the flavin (293) with the amines (294, 295) has been shown to yield a single photoproduct in each case identified as (296) and (297), respectively. 

Other light-absorbing trace organic compounds such as flavins, as well as inorganic compounds such as nitrate, nitrite, and metal complexes, do not contribute significantly to the total water column absorption. However, many of these compounds are quite photoreactive and will undergo rapid transformation under appropriate light fields. 

Figure 11 shows the result of this experiment on a solution of 5 mM N-acetyl tryptophan and 0.2 mM 3-N-carboxy-methyl lumiflavin, hereafter simply called flavin (see Figure 10). Positive enhancements can be observed for the aromatic C-2, C-4 and C-6 protons, while the CH2 group shows emission. This polarization pattern corresponds with a tryptophyl radical in which the electron spin is delocalized over the aromatic ring. It can further be noted that almost no flavin polarization is present in the difference spectrum. Figure 11c (weak lines are present at 2.6 and 4.0 ppm). This is due to cancellation of recombination and escape polarization as will be discussed in Section 5. The mechanism of the photoreaction undoubtedly involves triplet flavin (17). Since 1-N-methyl tryptophan shows similar CIDNP effects, the primary step most probably is electron transfer to the photo-excited flavin. This is also supported by a flash photolysis study by Heelis and Phillips (18). The nature of the primary step in the photoreactions with amino acids is important in view of the interpretation of "accessibility" of an amino acid side chain in a protein as seen by the photo-CIDNP method. This question is therefore the subject of further study.

In contrast to the case of tryptophan the photoreactions with tyrosine and histidine probably involve hydrogen atom transfer as the primary step. There are several indications for this. First, 0-methylated tyrosine (p-methoxy phenylalanine) did not show any photo-CIDNP effect and its reactivity as a photo-reductant towards flavins is strongly reduced (19). Similarly, 1-N-methyl histidine is not polarized at high pH (> 7.5), when no abstractable hydrogen is present. Secondly, in the protein ribonuclease A, which has a well known 3-dimensional structure, the residues Tyr 92 and His 105 have exposed rings, but their OH and NH protons are hydrogen bonded to backbone carbonyl groups. 

These residues do not show CIDNP, whereas several other residues do (Tyr 76, Tyr 115, His 119) (20). This strongly suggests that in the cases of tyrosine and histidine free phenolic OH and imidazole NH protons are required for the photoreaction with flavin in accordance with the hydrogen atom transfer mechanism. 

Two mesoheme and one FAD moieties are present within a polypeptide of molecular weight about 72,000 (Bartsch, 1968). The biological function of the cytochrome has not been conclusively demonstrated, but it has been implicated as an electron transfer intermediary between reduced sulfur substrates and the photoreaction center. This role has been demonstrated for an analogous monoheme flavin containing cytochrome c from C. thiosulphatophiliwn (Kusai and Yamanaka, 1973). 

Finally, two very special flavin-dependent photoreactions need to be mentioned ... 

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