An FMN-containing NADH-quinone reductase from Streptomyces sp.

NADH-quinone reductase was purified 22-fold from the cytosolic fraction of Streptomyces sp. IMSNU-1 to apparent homogeneity, with an overall yield of 9%, by the purification procedure consisting of ammonium sulfate precipitation and DEAE Sephacel, Sephacryl S-200 and DEAE 5 PW chromatographies. The molecular mass of the enzyme determined by gel filtration chromatography was found to be 110 kDa. SDS-PAGE revealed that the enzyme consists of two subunits with a molecular mass of 54 kDa. The enzyme contained 1 mol of FMN per subunit as a cofactor. The A(272)/(457) ratio was 6.14 and the molar extinction coefficients were calculated to be 20, 800 and 25,400 M(-1)cm(-1) at 349 and 457 nm, respectively. The N-terminal sequence of the enzyme contained the highly conserved fingerprint of ADP-binding domain. The enzyme used NADH as an electron donor and various quinones as electron accepters. Cytochrome c was practically inactive. Air-stable flavin semiquinone was produced by the addition of NADH to the enzyme. Also, naphthosemiquinone was detected in the reaction mixture containing the enzyme.