Preparation of [Fe]-Hydrogenase from Methanogenic Archaea

[Fe]-hydrogenase is one of the three types of hydrogenases. This enzyme is found in many hydrogenotrophic methanogenic archaea and catalyzes the reversible hydride transfer from H 2 to methenyl-H 4MPT + in methanogenesis from H 2 and CO 2. The enzyme harbors a unique iron-guanylyl pyridinol (FeGP) cofactor as a prosthetic group. Here, we describe the purification of [Fe]-hydrogenase from Methanothermobacter marburgensis, the isolation of the FeGP cofactor from the native holoenzyme, and the reconstitution of [Fe]-hydrogenase from the isolated FeGP cofactor and the heterologously produced apoenzyme.