Evidence for a Novel Flavin Prosthetic Group Associated with Nadh Dehydrogenase From

I. NADH dehydrogenase (EC 1.6.99.3) has been purified from the strictly anaerobic rumen bacterium Peptostreptococcus elsdenii. The purified protein is specific for NADH as electron donor; it can use 2,6-dichlorophenolindophenol (DCIP), cytochrome c, KzFe(CN)6 or flavodoxin as electron acceptor. It has a molecular weight of about 63 ooo. The absorption spectrum shows maxima at 475, 375, 356, 318, and 273 nm. I t has shoulders at 425,460, and 51o nm. The protein is fuorescent with an emission maximum at 550 nm. 2. The chromophore is released from the NADH dehydrogenase by either heat treatment or extraction with trichloroacetic acid. The free chromophore shows an increase of absorption in the visible spectrum and a shift of the maximum to 47 ° nm. The fuorescence emission maximum is shifted to 530 nm, but the intensity is unchanged. 3Hydrolysis of the free chromophore with phosphodiesterase causes a 12% increase in the visible absorption and a 2-fold increase in the fluorescence (exciting light at 480 nm). The chromatographic behavior is also affected by this treatment. 4. The hydrolysed chromophore is bound by apoflavodoxin with 95 % quenching of the fluorescence and an 18% decrease in the visible absorption. The complex of apoflavodoxin and the unknown chromophore is reduced by light irradiation in the presence of EDTA to an intermediate similar to flavodoxin semiquinone but with a distinctively different absorption spectrum. It is concluded that the chromophore in the NADH dehydrogenase is a flavin dinucleotide similar to FAD but modified in the isoalloxazine ring.