Purification and characterization of NADH dehydrogenase complex from Paracoccus denitrificans.

An NADH dehydrogenase complex was isolated from the plasma membranes of aerobically grown Paracoccus denitrificans cells by extraction with NaBr and purification on an NAD-agarose column. The NADH-ubiquinone-1 reductase activity of the isolated NADH dehydrogenase complex was about 10 times higher than that of the NaBr extract. The preparation was composed of 10 (6 major and 4 minor) unlike polypeptides, and lacked identifiable components and activities characteristic of other enzyme complexes of the oxidative phosphorylation system. The purified enzyme contained noncovalently bound FMN, nonheme iron, and acid-labile sulfide. The ratio of FMN to nonheme iron to acid-labile sulfide was 1:13 approximately 14:11 approximately 12, suggestive of the presence of multiple iron-sulfur clusters. The isolated NADH dehydrogenase complex cross-reacted with antisera to beef heart mitochondrial complex I and protein fraction derived therefrom, indicating the presence in the Paracoccus enzyme of antigenic sites similar to those in the intact complex I and its iron-sulfur protein and possibly hydrophobic protein fractions.