In this report the first high-quality infrared spectra of [Fe]-hydrogenase are presented. Analyses of these spectra obtained under a variety of redox conditions strongly indicate that [Fe]-hydrogenases contain a low-spin Fe ion in the active site with one CN Ϫ group and one CO molecule as intrinsic, non-protein ligands. When in the ferric state, the presence of such an ion can explain the enigmatic EPR properties (the rhombic 2.10 signal) of the active, oxidised enzyme. To account for other, well-characterised properties of the active site, we propose that the active site of [Fe] A third class of hydrogenase, not containing any metals and only active in the presence of its cofactor, has been discovered in methanogenic Archaea by Thauer and coworkers [8,9].A few [NiFe]-hydrogenases have been extensively studied. The basic unit of these enzymes is formed by a large (47Ϫ 72 kDa) and a small (23Ϫ38 kDa) subunit. In the last 4 years FTIR studies on the enzymes from Chromatium vinosum [10Ϫ 13] and Desulfovibrio gigas [14,15], combined with the crystal structure of the D. gigas enzyme [14,16] have revealed that the active site is a bimetallic NiFe site with two CN Ϫ groups and one CO molecule bound to Fe. The whole site is bound to the large subunit via four thiols from strictly conserved Cys residues. In the D. gigas enzyme, the small subunit harbours one [11,14,15]. Also nitrile hydratase exhibits an FTIR band in this region : in its inactive state, this enzyme has an NO bound to Fe giving rise to an FTIR band around 1855 cm Ϫ1 [17].In this study we have examined the FTIR bands exhibited by [Fe]-hydrogenase from D. vulgaris, strain Hildenborough, in more detail. Also the behaviour of the bands under various conditions was studied and compared with that of [NiFe]-hydrogenase from C. vinosum studied under the same conditions. It is concluded that the active site in [Fe]-hydrogenase contains a low-spin Fe with one CN Ϫ and one CO as intrinsic, non-protein ligands. The Fe ion can be either Fe 3ϩ , giving rise to the rhombic 2.10 EPR signal, or Fe 2ϩ (EPR silent). It is proposed that this low-spin Fe is directly linked to a [4Fe-4S] cluster, presumably via two bridging thiols from conserved Cys residues. MATERIALS AND METHODSGrowth of D. vulgaris, subspecies Hildenborough (NCIB 8303), purification of its [Fe]-hydrogenase and determination of purity and activity were performed as described previously [7]. The purity index (A 400 /A 280 ) of the preparation used was 0.36 and the H 2-production activity was 2255 U/mg. Growth of C. vinosum (DSM 185), purification of its [NiFe]-hydrogenase and activity measurements were as previously de-