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Energy-converting [NiFe] hydrogenases in archaea and bacteria: insights into the energy-transducing mechanism

By Lucia Forzi


In recent years a group of multisubunit membrane-bound [NiFe] hydrogenases has been identified in a variety of anaerobic or facultative anaerobic microorganisms. These enzymes share two conserved integral membrane proteins and four conserved hydrophilic proteins with the energy-conserving NADH:quinone oxidoreductases (complex I). Based on experimental evidence derived from physiological and biochemical studies, the various members of this hydrogenase family have been proposed to function as ion pumps being involved in energy-conserving electron transport, reverse electron transport, or both. Therefore these enzymes have been designated energy-converting [NiFe] hydrogenases. In the present work the energy transducing mechanism of energy-converting hydrogenases was studied in comparison to complex I. A particular attention was given to the prosthetic groups involved in the electron transfer pathway, the role of the membrane part and the identification of the coupling ion used by these enzymes. The majority of the experiments were carried out with Ech hydrogenase from Methanosarcina barkeri. The sequence of Ech hydrogenase predicts the binding of three [4Fe-4S] clusters, one by subunit EchC and two by subunit EchF. Previous studies had shown that two of these clusters could be fully reduced under 1 bar of H2 at pH 7 giving rise to two distinct S ½ EPR signals, designated as the g = 1.89 and the g = 1.92 signal. Redox titrations at different pH values demonstrated that these two clusters had a pH-dependent midpoint potential indicating a function in ion pumping. To assign these EPR signals to the subunits of the enzyme a set of M. barkeri mutants was generated in which seven of eight conserved cysteine residues in EchF were individually replaced by serine. EPR spectra recorded from the isolated mutant enzymes revealed a strong reduction or complete loss of the g = 1.92 signal whereas the g =1.89 signal was still detectable as the major EPR signal in five mutant enzymes. It is concluded that the cluster giving rise to the g = 1.89 signal is the proximal cluster located in EchC and that the g = 1.92 signal results from one of the clusters of subunit EchF. The pH-dependent midpoint potential of these two [4Fe-4S] clusters suggests that these clusters simultaneously mediate electron and ion transfer and thus could be an essential part of the ion-translocating machinery. In the two integral membrane subunits of Ech carboxylic residues are found that are highly conserved within the family of energy-converting hydrogenases and complex I. These residues could be part of a transmembrane ion channel. In line with this, Ech hydrogenase activity was inhibited by the carboxyl-modifying reagent N,N’–dicyclohexylcarbodiimide (DCCD). The inhibition of the enzyme correlated quite well with the incorporation of [14C]DCCD in subunits EchA. Using a combination of FT-IR difference spectroscopy and electrochemistry it was shown that the electron transfer reaction catalyzed by Ech hydrogenase from M. barkeri induces a conformational change of the enzyme and the protonation of amino acid side chains. Oxidized minus reduced spectra in the mid infrared range (1800 to 1200 cm-1) revealed conformational changes in the amide I region and a signal at 1720 cm-1 attributed to either an Asp or Glu side chain, protonated in the oxidized state. To identify the coupling ion used by energy converting hydrogenases studies with the enzyme from Carboxydothermus hydrogenoformans were performed. Cell suspensions of C. hydrogenoformans were found to couple the oxidation of CO to CO2 and H2 with the translocation of protons across the membrane at pH 5.9. This transient acidification was inhibited by the protonophore CCCP but was not affected by the sodium ionophore ETH-157, indicating the generation of a primary electrochemical proton gradient. However, no proton translocation coupled to CO oxidation was observed at pH 6.7. On the other hand, at neutral pH, CO oxidation was coupled to sodium ion translocation. This reaction was protonophore insensitive, indicating a primary Na+ translocation. These data indicate that the Coo hydrogenase from C. hydrogenoformans could be a primary sodium pump, which may also use H+ at low pH

Topics: NADH:Qoxidoreductase ; Iron-sulfur proteins ; Complex I ; Methanogenic archaea ; [NiFe] hydrogenases ; Archaebakterien ; Hydrogenasen ; Life sciences, NADH:Qoxidoreductase -- Iron-sulfur proteins -- Complex I -- Biowissenschaften, Biologie -- Methanogenic archaea -- [NiFe] hydrogenases -- Archaebakterien -- Hydrogenasen, ddc:570
Publisher: Philipps-Universität Marburg, Fachbereich Biologie
Year: 2005
OAI identifier:

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