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.