Zu Komplex I verwandte Hydrogenasen in Carboxydothermus hydrogenoformans und Thermoanaerobacter tengcongensis

From the membrane fraction of the Gram-positive bacterium Carboxydothermus hydrogenoformans, an enzyme complex catalyzing the conversion of CO to CO2 and H2 was purified. The enzyme complex showed maximal CO-oxidizing:H2-evolving enzyme activity with 5% CO in the headspace (450 U per mg protein). Higher CO concentrations inhibited the hydrogenase present in the enzyme complex. For maximal activity, the enzyme complex had to be activated by either CO or strong reductants. The enzyme complex also catalyzed the CO- or H2-dependent reduction of methylviologen at 5900 and 180 U per mg protein, respectively. The complex was found to be composed of six hydrophilic and two hydrophobic polypeptides. The amino-terminal sequences of the six hydrophilic subunits were determined allowing the identification of the encoding genes in the preliminary genome sequence of C. hydrogenoformans. From the sequence analysis it was deduced that the enzyme complex is formed by a Ni-containing carbon monoxide dehydrogenase (CooS), an electron transfer protein containing four [4Fe4S] clusters (CooF) and a membrane bound [NiFe] hydrogenase composed of four hydrophilic subunits and two membrane integral subunits. The hydrogenase part of the complex shows high sequence similarity to members of a small group of [NiFe] hydrogenases with sequence similarity to energy conserving NADH:quinone oxidoreductases. The data support a model in which the enzyme complex is composed of two catalytic sites, a CO-oxidizing site and a H2-forming site, which are connected via a different ironsulfur cluster containing electron transfer subunits. The exergonic redox reaction catalyzed by the enzyme complex in vivo has to be coupled to energy conservation, most likely via the generation of a proton motive force. Thermoanaerobacter tengcongensis is a thermophilic Gram-positive bacterium able to dispose of the reducing equivalents generated during the fermentation of glucose to acetate and CO2 by reducing H+ to H2. A unique combination of hydrogenases, a ferredoxin-dependent [NiFe] hydrogenase and an NADH-dependent Fe-only hydrogenase, were found to be responsible for H2 formation in this organism. Both enzymes were purified and characterized. The tightly membrane-bound [NiFe] hydrogenase belongs to a small group of complex-I-related [NiFe] hydrogenases and has highest sequence similarity to energy-converting [NiFe] hydrogenase (Ech) from Methanosarcina barkeri. A ferredoxin isolated from Ta. tengcongensis was identified as the physiological substrate of this enzyme. The heterotetrameric Fe-only hydrogenase was isolated from the soluble fraction. It contained FMN and multiple iron–sulfur clusters, and exhibited a typical H-cluster EPR signal after autooxidation. Sequence analysis predicted and kinetic studies confirmed that the enzyme is an NAD(H)-dependent Fe-only hydrogenase. When H2 was allowed to accumulate in the culture, the fermentation was partially shifted to ethanol production. In cells grown at high hydrogen partial pressure [p(H2)] the NADH-dependent hydrogenase activity was fourfold lower than in cells grown at low p(H2), whereas aldehyde dehydrogenase and alcohol dehydrogenase activities were higher in cells grown at elevated p(H2). These results indicate a regulation in response to the p(H2).