Studies on the Reaction Mechanisms of Flavoenzymes by Multi-Nuclear NMR

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1. D-AMINO ACID OXIDASE(DAO)Substrate analogs, o-, m-, and p-flucrobenzoate were used for elucidating the electron transfer from a substrate to oxidized flavin by means of F(19)NMR. Of the NMR signals of these analogs, the signal of o-derivative exhibited the largest chemical shift change when bound to DAO, while those ofm- and p-derivativesexhibitedmoderateandminorchanges, respectively. This is consistent with the electron transfer from the amino nitrogen of a substrate to the oxidized flavin.NADPH-ADRENODOXIN REDUCTASE(ADR)ADR was reconstituted with C(13)-enriched FAD and the C- 13 NMR spectra of the reconstituted ADR in the free state and in the complexed state with NADP were measured. The positions enriched were 2, 4, 4a, and loa-positions of the flavin nucleus. In the complexed form with NADP, the 4a-signal-was down-field shifted, suggesting that when electrons are transferred from NADPH to oxidized flavin the NADPH-binding lowers the electron density at the 4a-position thereby facilitating the electron transfer.3. OLD YELLOW ENZYME(OYE)When OYE reconstituted with 8-fluoro-FMN was subjected to limited proteolysis by chymotrypsin, the protein was specificaly cleaved to yield 14KD and 34KD fragments. Subsequently, the amino group of the leucine residue of the N-terTninus of the 14KD fragment generated by the cleavage attacked the 8-position of 8-fluoro-FMN nucleophilically and a covalent bond was fontied between the flavin moiety and the 14KD fragment. From these results we could deduce the position of FMN binding and the mode of binding of FMN in OYF-