Most methanogenic archaea reduce CO(2) with H(2) to CH(4). For the activation of H(2), they use different [NiFe]-hydrogenases, namely energy-converting [NiFe]-hydrogenases, heterodisulfide reductase-associated [NiFe]-hydrogenase or methanophenazine-reducing [NiFe]-hydrogenase, and F(420)-reducing [NiFe]-hydrogenase. The energy-converting [NiFe]-hydrogenases are phylogenetically related to complex I of the respiratory chain. Under conditions of nickel limitation, some methanogens synthesize a nickel-independent [Fe]-hydrogenase (instead of F(420)-reducing [NiFe]-hydrogenase) and by that reduce their nickel requirement. The [Fe]-hydrogenase harbors a unique iron-guanylylpyridinol cofactor (FeGP cofactor), in which a low-spin iron is ligated by two CO, one C(O)CH(2)-, one S-CH(2)-, and a sp(2)-hybridized pyridinol nitrogen. Ligation of the iron is thus similar to that of the low-spin iron in the binuclear active-site metal center of [NiFe]- and [FeFe]-hydrogenases. Putative genes for the synthesis of the FeGP cofactor have been identified. The formation of methane from 4 H(2) and CO(2) catalyzed by methanogenic archaea is being discussed as an efficient means to store H(2).

a b s t r a c t[Fe]-hydrogenase is one of three types of enzymes known to activate H 2 . Crystal structure analysis recently revealed that its active site iron is ligated square-pyramidally by Cys176-sulfur, two CO, an ''unknown" ligand and the sp 2 -hybridized nitrogen of a unique iron-guanylylpyridinol-cofactor. We report here on the structure of the C176A mutated enzyme crystallized in the presence of dithiothreitol (DTT). It suggests an iron center octahedrally coordinated by one DTT-sulfur and one DTToxygen, two CO, the 2-pyridinol's nitrogen and the 2-pyridinol's 6-formylmethyl group in an acyliron ligation. This result led to a re-interpretation of the iron ligation in the wild-type.

An open and closed case: The structure of a binary complex of C176A [Fe]‐hydrogenase with methylenetetrahydromethanopterin was solved at 2.15 Å resolution in an open conformation. A closed form of the complex was modeled on the basis of the experimentally determined structure. In this model, the iron‐site trans to the acyl carbon is located next to the C14a and therefore considered as H2 binding site.

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