title: 8-Hydroxy-5-deazaflavin-reducing hydrogenase from Methanobacterium thermoautotrophicum: 1. Purification and characterization.
abstract: The 8-hydroxy-5-deazaflavin (coenzyme F420) reducing hydrogenase from the obligate anaerobe Methanobacterium thermoautotrophicum delta H has been purified 41-fold to apparent homogeneity. The major active enzyme form is a high molecular weight aggregate of Mr ca. 800,000, composed of three subunits, alpha (Mr 47K), beta (Mr 31K), and gamma (Mr 26K). The hydrogenase is purified aerobically in reversibly inhibited form, and conditions for anaerobic reductive activation with H2, high salt, thiols, and electron acceptors have been defined. The minimal species transferring electrons from H2 to coenzyme F420 appears to be an alpha beta delta (Mr 115K) complex. The tightly associated redox cofactors per 115K species are 0.6-0.7 nickel atom, 0.8-0.9 flavin adenine dinucleotide (FAD), and 13-14 iron atoms in iron-sulfur centers. The subunits have been separated by denaturing gel electrophoresis, which has permitted determination of amino acid composition, subunit N-terminal sequencing, and preparation of subunit-directed antibodies. There is iron associated with the alpha-subunit, but placement of the nickel and FAD has not been established.
authors: Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
authors: J A
authors: JA
authors: Fox
authors: D J
authors: DJ
authors: Livingston
authors: W H
authors: WH
authors: Orme-Johnson
authors: C T
authors: CT
authors: Walsh
mesh_terms: D000595:Amino Acid Sequence; D019605:Euryarchaeota / Q000201:enzymology*; D005486:Flavin Mononucleotide / Q000032:analysis; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D046911:Macromolecular Substances; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D010088:Oxidoreductases; C033895:coenzyme F420 hydrogenase