Preparation and some properties of 6-substituted flavins as active site probes for flavin enzymes

Abstract

6-Azidoflavins, 6-thiocyanatoflavins, and 6-mercaptoflavins at the lumiflavin, riboflavin, FMN, and FAD level were prepared from the corresponding 6-aminoflavins and some of their properties investigated. They are bound tightly by apoflavin enzymes which bind either riboflavin, FMN, or FAD. 6-Azidoflavins undergo facile photolysis. One major product was identified as 6-aminoflavin. A further product, which was formed also during acid decomposition of the azide, results from opening of the flavin benzene ring and is proposed to have a lumazine structure. 6-Thiocyanatoflavins are easily converted by dithiothreitol to 6-mercaptoflavins. The latter are stabilized against dimerization in the presence of reducing thiols. 6-Mercaptoflavins have a pK of 5.9, which corresponds to ionization of the 6-SH function. The neutral form is yellow, while the anion is green, due to a long-wavelength band ( A , , , -600 nm) extending beyond 700 nm. These properties suggest the use of these 6-substituted flavins for probing the active site of flavin enzymes. Because their reactive substituents are in close proximity to the flavin N(5)-position, these 6-substituted derivatives should also serve as useful probes of the environment around the flavin N(5), a position known to be involved in all flavin-mediated redox processes.

New flavins for old: artificial flavins as active site probes of flavoproteins

“…This should be contrasted with the effects observed upon binding of 8-mercaptoriboflavin to riboflavinbinding protein (Fig. 5), where the spectrum typical for form (a) is obtained, in agreement with the known preference of this protein for binding neutral flavins [21,34]. From these considerations it is apparent that 8-mercaptoflavins can yield very important information on the localization of charge dipoles/hydrogen bridges at the active centre of flavin enzymes.…”

“…This might be ascribed to the differences of sulphur and oxygen in undergoing if-interactions and in electronegativity. 6-Hydroxy- [18] and 6-mercaptoflavins [21] ( Fig. 6) can be described by a similar set of structures as the 8-substituted analogues (cf.…”

“…This enzyme has been purified recently to homogeneity (D. J. Manstein & E. F. Pai, unpublished work). In some cases, in which the enzyme would not accept riboflavin derivatives as substrates, the preparation of FAD analogues modified at position C (6) has been achieved by chemical conversions at the FAD level [21].…”

“…Recently we have also begun to study enzymes reconstituted with 6-thiocyanatoflavins and 6-mercaptoflavins (V. Massey, S. Ghisla & K. Yagi, unpublished work) [21]. These flavins and some of their properties had previously been reported in connection with the structural determination of the covalently bound flavin of trimethylamine dehydrogenase [50,51].…”

“…In contrast to the 8-azidoflavins the recently synthesized 6-azidoflavins are less light sensitive, thus simplifying their handling [21]. Their photoactivity, however, is sufficiently high as to yield appreciable covalent reaction with several proteins (V. Massey, S. Ghisla & K. Yagi, unpublished work).…”