Formation of epoxide intermediates in the reaction of enzyme-bound 5-deazaflavin with peroxides.

Reaction of free 5-deazaflavin with Hz02 or m-chloroperoxybenzoate results in the formation of 4a,5epoxy derivatives. These compounds (Arna 340 nm) are nonfluorescent, decompose in neutral aqueous solution, and react with iodide to regenerate the original parent compound. The initial reaction of peroxides with 5-deazaFAD bound to oxynitrilase or with 5-deazaFMN bound to glycolate oxidase results in the rapid formation of an intermediate (X,,,340 nm) which i s converted to enzyme-bound 5-deazaflavin-X via a slow, peroxide-independent pathway. DeazaFAD .X (A, = 343, 359 nm), isolated from oxynitrilase, exhibits an intense purple fluorescence (emission X, = 383 nm), is stable in aqueous solution, and does not react with iodide. DeazaFMNoX, prepared with glycolate oxidase or by treating deazaFAD-X with phosphodiesterase, can be tightly bound to apoflavodoxin (Kn = 9.3 m). The following results indicate that the initial intermediate in the nzyme reactions is an epoxide derivative. The same intermediate is formed with H202 or m-chloroperoxybenzoate. The latter reaction is accompanied by the formation of stoichiometric amounts of m-chlorobenzoate. The intermediate reacts with iodide to regenerate unmodified enzyme. The spectral properties of the intermediate and the decomposition observed for the protein-free intermediate are similar to that observed for the model epoxide compounds. The conversion of the epoxide intermediate to enzyme-bound 5deazaflavin .X is facilitated by the protein moiety since the reaction is nhibited by compounds which bind near the coenzyme site or prevented by enzyme denaturation. This conversion with glycolate oxidase proceeds via a series of two, irreversible reactions involving a second intermediate (Arnm 5: 355, 375 nm), which does not react with iodide and which was not seen in the case of oxynitrilase. Unlike glycolate oxidase and oxynitrilase, 5-deazaflavin bound to flavodoxin or to the riboflavin-binding protein neither reacts with peroxides nor forms covalent adducts with sulfite or cyanide. The results suggest that epoxidation of enzyme-bound 5-deazaflavin may be correlated with the susceptibility of the coenzyme toward nucleophilic attack at position 5.