A family of flavoproteins, called A-type flavoproteins, is described. It consists of 14 protein sequences of 385Ϫ597 amino acids in length, 7 from methanogens (domain: Archaea), 5 from phototrophic prokaryotes, one from Escherichia coli, and a partial sequence from the sulfate reducer Desulfovibrio gigas (domain : Bacteria). No similar sequence could be found in the domain Eucarya. All sequences show significant similarity over a 385Ϫ400 amino acid portion overlapping a recognizable flavodoxin signature starting at positions 245Ϫ285 of the common core sequence. Cofactor analysis and, to some extent, analysis of the primary structure of six A-type flavoproteins, three of which are structurally characterized here, support the existence of four sub-families: Here we have aligned 14 sequences and FAD, which enables them to catalyze a variety of oneelectron and two-electron transfer reactions [1]. A number of and characterized the R. capsulatus protein and 2 additional members of this group of proteins as simple or complex flavoflavoproteins are known that contain additional redox centers like a disulfide bond, a heme group, a metal, or iron-sulfur cen-proteins. As a result, we propose to group them into a heterogeneous family, the A-type flavoproteins. ters [2]. Flavoproteins known to bind several cofactors, like cytochromes P-450, have distinct binding sites often located on separate protein domains [3Ϫ5].EXPERIMENTAL PROCEDURES We have recently purified an FMN-containing flavoprotein from cells of Methanobacterium thermoautotrophicum, flavoMaterials, Chemicals and enzymes. Restriction enzymes, protein A, which, like flavodoxins, is up-regulated under iron T4 DNA ligase, and Taq polymerase were purchased from and limitation and possesses a perfectly conserved flavodoxin signa-used as recommended by Fermentas. Oligonucleotide primers ture [6]. However, it differs from typical flavodoxins in being were synthesized at Microsynth. Q Sepharose Fast Flow, Sephmuch larger (a dimer of a 404-amino-acid polypeptide) and in acryl S-200 and S-300 HR were from Pharmacia, and Fractogel having a relatively high redox potential of Ϫ230 mV for the EMD-TMAE(650)-S from Merck (Darmstadt, Germany). Other quinone/hydroquinone couple [7]; so far, its function remains chemicals were obtained from Fluka (Buchs, Switzerland), and hypothetical [6Ϫ7]. At the time of its discovery, there was only were from the highest grade available. one flavoprotein clearly homologous to M. thermoautotroSequence analysis. Sequence analyses were carried out with phicum flavoprotein A, namely the product of an open reading the University of Wisconsin's Genetics Computer Group packframe in an operon involved in nitrogen fixation in Rhodobacter age, versions 8 and 9 and at the National Center for Biotechnolcapsulatus [8Ϫ9]. Since then, the completion of several se-ogy Information via the BLAST network service. quencing projects has permitted the discovery of ten predicted Amplification, cloning, and expression of E. coli orf479, polypeptide sequences unambiguou...