Fusion of two subunits does not impair the function of a [NiFeSe]‐hydrogenase in the archaeon Methanococcus voltae

Abstract

[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...

Dimethylselenide Demethylation Is an Adaptive Response to Selenium Deprivation in the Archaeon Methanococcus voltae

“…A reporter gene construct was used to show this effect. M. voltae strain V1 carrying the Escherichia coli ␤-glucuronidase gene uidA under the control of the regulated vhc hydrogenase promoter (20) was grown under selenium limitation and shown to produce ␤-glucuronidase. Upon addition of dimethylselenide, the hydrogenase promoter was shut down, indicating that the internal selenium concentration necessary to affect the repression had been exceeded (Table 3).…”

The vhuU gene encoding a small subunit of a selenium-containing [NiFe]-hydrogenase in Methanococcus voltae appears to be essential for the cell

“…A role in the negative regulation of the frc and vhc transcription in the presence of selenium has been discussed (Sorgenfrei et al 1997). Recently, a mutant of M. voltae that carries a fusion of the vhuA gene and the vhuU gene to form a composite subunit equivalent (VhuF) to the large subunits of the common [NiFe]-hydrogenases of other prokaryotes has been constructed (Pfeiffer et al 1998). This mutant is viable when grown under selenium depletion, although in this mutant the free vhuU gene product is no longer available and still shows the normal transcriptional regulation of the genes encoding the selenium-free [NiFe]-hydrogenases.…”

“…8. As controls we included wild-type DNA and DNA from a transformant carrying a fusion of genes vhuA and vhuU, named vhuF, which results in the synthesis of a functional fusion subunit, VhuF (Pfeiffer et al 1998). This transformant had been produced using an identical vector, except that it contained the vhuF gene instead of the vhuA gene followed by the vhuU deletion.…”

“…A Assumed mode of integration into a replication fork and asymmetric recombination, followed by the separation of the daughter chromosomes. The chromosomal configuration of the control carrying the vhuF fusion gene after gene replacement (Pfeiffer et al 1998) is also shown. The shaded symbols show the pac cassette flanked by the Pmcr promoter (triangle) and the Tmcr terminator (diamond).…”

Nickel–Iron–Selenium Hydrogenases – An Overview

“…These two subunits are encoded by the vhuA and vhuU genes that probably resulted from a gene splitting event. The fusion of the vhuA and vhuU genes has no effect in the activity of the enzyme [30] and the small fragment is believed to be important in the expression regulation of these Hases [28] . VhuG is the small electron transferring subunit, and VhuD mediates the electron transfer between the VhuG and the Hdr [2b, 27] .…”