Membrane-bound NiFe Hydrogenase Research Articles

Purification and molecular characterization of the H2 uptake membrane-bound NiFe-hydrogenase from the carboxidotrophic bacterium Oligotropha carboxidovorans.

The membrane-bound hydrogenase of Oligotropha carboxidovorans was solubilized with n-dodecyl-beta-D-maltoside and purified 28-fold with a yield of 29% and a specific activity of 173 to 178 micromol of H2 x min(-1) x mg(-1). It is the first hydrogenase studied in a carboxidotrophic bacterium. The enzyme acts on artificial electron-accepting dyes, such as methylene blue, but is ineffective with pyridine nucleotides or other soluble physiological electron acceptors. Hydrogenase of O. carboxidovorans belongs to class I of hydrogenases and is a heterodimeric 101,692-Da NiFe-protein composed of the polypeptides HoxL and HoxS. Molecular cloning data revealed, that HoxL comprises 604 amino acid residues and has a molecular mass of 67,163 Da. Pre-HoxS comprises 360 amino acid residues and is synthesized as a precursor protein which is cleaved after alanine at position 45, thus producing a mature HoxS of 33,767 Da. The leader sequence corresponds to the signal peptide of small subunits of hydrogenases. The hydropathy plots of HoxL and HoxS were indicative for the absence of transmembranous helices. HoxZ has four transmembranous helices and is considered the potential membrane anchor of hydrogenase in O. carboxidovorans. Hydrogenase genes show the transcriptional order 5' hoxV --> hoxS --> hoxL --> hoxZ 3'. The hox gene cluster as well as the clustered CO dehydrogenase (cox) and Calvin cycle (cbb) genes are arranged within a 30-kb DNA segment of the 128-kb megaplasmid pHCG3 of O. carboxidovorans.

Requirement for nickel of the transmembrane translocation of NiFe-hydrogenase 2 in Escherichia coli

The cellular location of membrane-bound NiFe-hydrogenase 2 (HYD2) from Escherichia coli was studied by immunoblot analysis and by activity staining. Treatment of spheroplasts with trypsin was able to release active HYD2 into the soluble fraction, indicating that HYD2 is attached to the periplasmic side of the cytoplasmic membrane and that HYD2 undergoes a trans-membrane translocation during its biosynthesis. By using a nik mutant deficient in the high affinity specific nickel transport system, we show that the intracellular availability of nickel is essential for the processing of the large subunit and for the transmembrane translocation of HYD2. We also demonstrate that the processing of the precursor, which is related with nickel incorporation, can occur in the membrane-depleted soluble fraction and that it is associated with the increase in resistance to proteolysis of the processed form of the large subunit. The mechanism of the transmembrane translocation of HYD2 is discussed.

Microbial hydrogenases: Primary structure, classification, signatures and phylogeny

Thirty sequenced microbial hydrogenases are classified into six classes according to sequence homologies, metal content and physiological function. The first class contains nine H 2-uptake membrane-bound NiFe-hydrogenases from eight aerobic, facultative anaerobic and anaerobic bacteria. The second comprises four periplasmic and two membrane-bound H 2 -uptake NiFe(Se)-hydrogenases from sulphate-reducing bacteria. The third consists of four periplasmic Fe-hydrogenases from strict anaerobic bacteria. The fourth contains eight methyl-viologen- (MV), factor F420- (F420) or NAD-reducing soluble hydrogenases from methanobacteria and Alcaligenes eutrophus H16. The fifth is the H 2-producing labile hydrogenase isoenzyme 3 of Escherichia coli. The sixth class contains two soluble tritium-exchange hydrogenases of cyanobacteria. The results of sequence comparison reveal that the 30 hydrogenases have evolved from at least three different ancestors. While those of class I, II, IV and V hydrogenases are homologous, i.e. sharing the same evolutionary origin, both class III and VI hydrogenases are neither related to each other nor to the other classes. Sequence comparison scores, hierarchical cluster structures and phylogenetic trees show that class II falls into two distinct clusters composed of NiFe- and NiFeSe-hydrogenases, respectively. These results also reveal that class IV comprises three distinct clusters: MV-reducing, F420-reducing and NAD-reducing hydrogenases. Specific signatures of the six classes of hydrogenases as well as some subclusters have been detected. Analyses of motif compositions indicate that all hydrogenases, except those of class VI, must contain some common motifs probably participating in the formation of hydrogen activation domains and electron transfer domains. The regions of hydrogen activation domains are highly conserved and can be divided into two categories. One corresponds to the ‘nickel active center’ of NiFe(Se)-hydrogenases. It consists of two possible specific nickel-binding motifs, RxCGxCxxxH and DPCxxCxxH, located at the N- and C-termini of so-called large subunits in the dimeric hydrogenases, respectively. The other is the H-cluster of the Fe-hydrogenases. It might comprise three motifs on the C-terminal half of the large subunits. However, the motifs corresponding to the putative electron transfer domains, as well as their polypeptides chains, are poorly or even not at all conserved. They are present essentially on the small subunits in NiFe-hydrogenases. Some of these motifs resemble the typical ferredoxin-like Fe-S cluster binding site. The variation of the sequences in these regions might determine the specific interaction between the external electron transfer domains of hydrogenases and their electron carriers. For example, the C-terminal cysteine-histidine rich region of the small subunits are distinguishable between those of H 2-uptake, H 2-producing, MV-reducing, F420-reducing and NAD-reducing NiFe(Se)-hydrogenases.

Microbial hydrogenases: primary structure, classification, signatures and phylogeny.

Thirty sequenced microbial hydrogenases are classified into six classes according to sequence homologies, metal content and physiological function. The first class contains nine H2-uptake membrane-bound NiFe-hydrogenases from eight aerobic, facultative anaerobic and anaerobic bacteria. The second comprises four periplasmic and two membrane-bound H2-uptake NiFe(Se)-hydrogenases from sulphate-reducing bacteria. The third consists of four periplasmic Fe-hydrogenases from strict anaerobic bacteria. The fourth contains eight methyl-viologen- (MV), factor F420- (F420) or NAD-reducing soluble hydrogenases from methanobacteria and Alcaligenes eutrophusH16. The fifth is the H2-producing labile hydrogenase isoenzyme 3 of Escherichia coli. The sixth class contains two soluble tritium-exchange hydrogenases of cyanobacteria. The results of sequence comparison reveal that the 30 hydrogenases have evolved from at least three different ancestors. While those of class I, II, IV and V hydrogenases are homologous, i.e. sharing the same evolutionary origin, both class III and VI hydrogenases are neither related to each other nor to the other classes. Sequence comparison scores, hierarchical cluster structures and phylogenetic trees show that class II falls into two distinct clusters composed of NiFe- and NiFeSe-hydrogenases, respectively. These results also reveal that class IV comprises three distinct clusters: MV-reducing, F420-reducing and NAD-reducing hydrogenases. Specific signatures of the six classes of hydrogenases as well as some subclusters have been detected. Analyses of motif compositions indicate that all hydrogenases, except those of class VI, must contain some common motifs probably participating in the formation of hydrogen activation domains and electron transfer domains. The regions of hydrogen activation domains are highly conserved and can be divided into two categories. One corresponds to the 'nickel active center' of NiFe(Se)-hydrogenases. It consists of two possible specific nickel-binding motifs, RxCGxCxxxH and DPCxxCxxH, located at the N- and C-termini of so-called large subunits in the dimeric hydrogenases, respectively. The other is the H-cluster of the Fe-hydrogenases. It might comprise three motifs on the C-terminal half of the large subunits. However, the motifs corresponding to the putative electron transfer domains, as well as their polypeptides chains, are poorly or even not at all conserved. They are present essentially on the small subunits in NiFe-hydrogenases. Some of these motifs resemble the typical ferredoxin-like Fe-S cluster binding site.(ABSTRACT TRUNCATED AT 400 WORDS)