Acidianus ambivalens type-II NADH dehydrogenase: genetic characterisation and identification of the flavin moiety as FMN

The thermoacidophilic archaeon Acidianus ambivalens contains a monomeric 47 kDa type-II NADH dehydrogenase (NDH), which contains a covalently bound flavin. In this work, by a combination of several methods, namely (31)P-nuclear magnetic resonance and fluorescence spectroscopies, it is proven that this enzyme contains covalent FMN, a novelty among this family of enzymes, which were so far thought to mainly have the flavin dinucleotide form. Discrimination between several possible covalent flavin linkages was achieved by spectral and fluorescence experiments, which identified an 8alpha-N(1)-histidylflavin-type of linkage. Analysis of the gene-deduced amino acid sequence of type-II NDH showed no transmembranar helices and allowed the definition of putative dinucleotide and quinone binding motifs. Further, it is suggested that membrane anchoring can be achieved via amphipatic helices.