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(4)MPT(+) 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.