As in many other hydrogenases, the small subunit of the F 420 -reducing hydrogenase of Methanococcus voltae contains three iron-sulfur clusters. The arrangement of the three [4Fe±4S] clusters corresponds to the arrangement of [Fe-S] Hydrogenases catalyze the reversible cleavage of the dihydrogen molecule into two protons and two electrons. They are found in organisms belonging to each of the three kingdoms, Archaea, Bacteria and Eukarya [1±6] where they play different roles. In the case of fermenting micro-organisms growing under anaerobic conditions, electrons are waste products which accumulate, e.g. on formation of organic acids as the result of abstraction of electrons from intermediate metabolites. On combination with protons, catalyzed by hydrogenases, hydrogen is formed, which can be excreted [7]. With the help of uptake hydrogenases, it can be used as a source of reducing equivalents by hydrogen-consuming organisms.Cleavage of the dihydrogen molecule occurs heterolytically at metal centers of the hydrogenases. Two alternative types of such centers have been described in different hydrogenases. One contains only Fe. The three-dimensional structure of a second atomic structure of an Fe-only hydrogenase has been published [8]. The other type contains a bimetallic [NiFe] center [9±11]. In both cases, diatomic CN and CO ligands are attached to the Fe atoms. Their functions remain unknown.In most cases, the bimetallic reaction center is co-ordinated by four cysteine ligands. In several instances, one of these residues, which are conserved as parts of the largest common subunit of almost all [NiFe] hydrogenases, is replaced by a selenocysteinyl residue [12±15]. The selenium atom is a ligand to the Ni atom and probably has a direct role in the hydrogen cleavage reaction [10].We are interested in structure-function relationships of such [NiFeSe] hydrogenases and have focused our studies on two enzymes from Methanococcus voltae. The two hydrogenases, Vhu and Fru, probably serve in the uptake of hydrogen in the methanogenic archaeon M. voltae, which generates its energy by the reduction of carbon dioxide with hydrogen [16,17]. The natural electron acceptor of the Vhu hydrogenase is not known. The electron acceptor of Fru is a deazaflavin, F 420 [18]. This enzyme consists of three subunits and contains FAD [3]. In addition to its proposed role as uptake hydrogenase, Fru also acts as an F 420 H 2 -dehydrogenase, generating electrons used for the terminal reductive step of methanogenesis [19].In [NiFe] hydrogenases, the bimetallic center catalyzes the dihydrogen cleavage. In the first step, a metal-bound hydride and a proton are formed. The electrons are then abstracted from the hydride and transferred to an electron acceptor. The electron transfer to the acceptor occurs through a different subunit of the enzyme which contains three linearly arranged evenly spaced [Fe±S]

[NiFe]-hydrogenases generally carry the bimetallic Ni-Fe reaction center on their largest subunit. The [NiFeSe]-hydrogenase Vhu from Methanococcus voltae has an unusual subunit composition. Some of the amino acids participating in the formation of the reaction center are within a separate, very small subunit, called VhuU. It consists of only 25 amino acids and contains the selenocysteinyl residue, a ligand to the Ni atom. We have tested whether the special configuration of the Vhu-hydrogenase is of particular biochemical relevance. We have constructed a fusion subunit derived from the VhuA and VhuU subunits by generating a gene fusion which was inserted into the chromosome of M. voltae by gene replacement. The enzyme was purified and shown to be as active as the wild-type enzyme. M. voltae carries the genetic information for four different [NiFe]-hydrogenases. In addition to the Vhu-hydrogenase, a second selenium-containing enzyme, Fru, has been purified. Two selenium-free enzymes, Vhc and Frc, are homologues of Vhu and Fru, respectively. Their gene groups, vhc and frc are transcribed only upon selenium depletion. The selenium-containing subunit VhuU has been implicated in their negative regulation. However, cells containing the fusion hydrogenase still exhibited normal regulation of the vhc and frc promoter activities as tested in reporter gene constructs. This indicates that the free VhuU polypeptide is not required for the negative regulation of the vhc or frc genes. second subunit. These two subunits form a functional enzyme as tested with artificial electron acceptors.Two [NiFeSe]-hydrogenases have been purified from the archaeon Methanococcus voltae. They have been characterized with respect of the electronic configurations of their primary reaction centers at different redox states [14Ϫ16]. One of these enzymes, termed Fru, reduces the deazaflavine cofactor F 420. It consists of three subunits with the canonical large subunit carrying the [NiFe] cluster. The other, Vhu, whose natural electron acceptor remains unknown, has a special subunit composition. While the subunit containing the Fe-S clusters corresponds to those of other [NiFe]-hydrogenases, the [NiFe] center is formed by two subunits each contributing two ligands [12,13]. It appears that the gene normally encoding the largest subunit has been split. The homologue of the larger N-terminal part of the subunit carrying the primary reaction center is encoded by the gene vhuA, directly followed by a very small gene vhuU encoding what would be the C-terminal part of a normal large subunit [14,17]. The vhuU gene product is C-terminally processed, probably prior to its incorporation into the enzyme and the formation of the [NiFe] cluster to which it contributes a cysteinyl residue and a selenocysteinyl residue. This processing is a general feature and normally occurs at the C-terminus of the large subunit polypetide concomitantly with the incorporation of Ni [18].M. voltae carries genetic information for two further [NiFe]-hydrogenases, Frc and Vhc, wh...

In Methanococcus voltae, one of the two [NiFeSe] hydrogenases is unusual in that the large subunit is split into two subunits, each contributing two ligands to the [NiFe] center that catalyzes the heterolytic cleavage of the dihydrogen molecule. We have engineered a fusion of these two subunits. The resulting new enzyme showed no significant difference in hydrogen uptake activity or in the Ni-C or Ni-L EPR spectra compared to the the wild-type enzyme, but exhibited a tenfold increase in both the Km for hydrogen and the Ki for the competitive inhibitor carbon monoxide.