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6-Thiocyanatoflavins and 6-mercaptoflavins as active-site probes of flavoproteins

6-Thiocyanatoflavins and 6-mercaptoflavins as active-site probes of flavoproteins

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MASSEY, Vincent, Sandro GHISLA, Kunio YAGI, 1986. 6-Thiocyanatoflavins and 6-mercaptoflavins as active-site probes of flavoproteins. In: Biochemistry. 25(24), pp. 8103-8112. ISSN 0006-2960. eISSN 1520-4995. Available under: doi: 10.1021/bi00372a046

@article{Massey19866Thio-8234, title={6-Thiocyanatoflavins and 6-mercaptoflavins as active-site probes of flavoproteins}, year={1986}, doi={10.1021/bi00372a046}, number={24}, volume={25}, issn={0006-2960}, journal={Biochemistry}, pages={8103--8112}, author={Massey, Vincent and Ghisla, Sandro and Yagi, Kunio} }

application/pdf 2011-03-24T17:41:38Z 1986 Massey, Vincent Ghisla, Sandro 2011-03-24T17:41:38Z eng First publ. in: Biochemistry 25 (1986), 24, pp. 8103-8112 6-Thiocyanatoflavins and 6-mercaptoflavins as active-site probes of flavoproteins Ghisla, Sandro Massey, Vincent Attribution-NonCommercial-NoDerivs 2.0 Generic Yagi, Kunio 6-Thiocyanatoflavins have been found to be susceptible to nucleophilic displacement reactions with sulfite and thiols, yielding respectively the 6-S-S03--flavin and 6-mercaptoflavin, with rate constants at pH 7.0, 20°C, of 55 M-1 min-1 for sulfite and 1000 M-1 min-1 for dithiothreitol. The 6-SCN-flavin binds tightly to riboflavin-binding protein as the riboflavin derivative, to apoflavodoxin, apo-lactate oxidase, and apo-Old Yellow Enzyme as the FMN derivative, and to apo-D-amino acid oxidase as the FAD derivative. The riboflavin-binding protein derivative is inaccessible to dithiothreitol attack, and the lactate oxidase and D-amino acid oxidase derivatives show only limited accessibility. However, the flavodoxin and Old Yellow Enzyme derivatives react readily with dithiothreitol, indicating that the flavin 6-position is exposed to solvent in these proteins. The lactate oxidase and D-amino acid oxidase derivatives convert slowly but spontaneously to the 6-mercaptoflavin enzyme forms in the absence of any added thiol, indicating the presence of a thiol residue in the flavin binding site of these proteins. The reaction rates have been investigated of 6-mercaptoflavins with iodoacetamide, N-ethylmaleimide, methyl methanethiolsulfonate, H202, and mchloroperbenzoate, in both the free and protein-bound state. The results confirm the conclusions drawn from the studies with 6-SCN-flavins described above and from 6-N3-flavins [Massey, V., Ghisla, S., & Yagi, K. (1986) Biochemistry (preceding paper in this issue)]. The spectral properties of the protein-bound 6-mercaptoflavin vary widely among the five proteins studied and show stabilization of the neutral flavin with flavodoxin and riboflavin-binding protein and of the anionic species by Old Yellow Enzyme, lactate oxidase, and D-amino acid oxidase. In the case of the latter two enzymes, the stabilization appears to be due to interaction of the negatively charged flavin with a positively charged protein residue located near the flavin pyrimidine ring. This positively charged residue appears to be responsible also for the strong stabilization of the two-electron oxidation state of the mercaptoflavin as the 6-S-oxide. With the other flavoproteins studied this oxidation level is stabilized as the 6-sulfenic acid or 6-sulfenate. Yagi, Kunio

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