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⁻ 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]‐hydrogenases consists of this low‐spin Fe ion bound to an unusual [4Fe‐4S] cluster via bridges with sulphur atoms.

ortho-Chlorophenol reductive dehalogenase of the halorespiring Gram-positiveDesulfitobacterium dehalogenans was purified 90-fold to apparent homogeneity. The purified dehalogenase catalyzed the reductive removal of a halogen atom from the ortho position of 3-chloro-4-hydroxyphenylacetate, 2-chlorophenol, 2,3-dichlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, pentachlorophenol, and 2-bromo-4-chlorophenol with reduced methyl viologen as electron donor. The dechlorination of 3-chloro-4-hydroxyphenylacetate was catalyzed by the enzyme at a V _max of 28 units/mg protein and a K _m of 20 μm. The pH and temperature optimum were 8.2 and 52 °C, respectively. EPR analysis indicated one [4Fe-4S] cluster (midpoint redox potential (E _m) = −440 mV), one [3Fe-4S] cluster (E _m = +70 mV), and one cobalamin per 48-kDa monomer. The Co(I)/Co(II) transition had an E _m of −370 mV. Via a reversed genetic approach based on the N-terminal sequence, the corresponding gene was isolated from a D. dehalogenans genomic library, cloned, and sequenced. This revealed the presence of two closely linked genes: (i) cprA, encoding the o-chlorophenol reductive dehalogenase, which contains a twin-arginine type signal sequence that is processed in the purified enzyme; (ii) cprB, coding for an integral membrane protein that could act as a membrane anchor of the dehalogenase. This first biochemical and molecular characterization of a chlorophenol reductive dehalogenase has revealed structural resemblance with haloalkene reductive dehalogenases.