Abstract
The oxygen-tolerant membrane-bound [NiFe]-hydrogenase (MBH) from Ralstonia eutropha H16 consists of three subunits. The large subunit HoxG carries the [NiFe] active site, and the small subunit HoxK contains three [FeS] clusters. Both subunits form the so-called hydrogenase module, which is oriented toward the periplasm. Membrane association is established by a membrane-integral cytochrome b subunit (HoxZ) that transfers the electrons from the hydrogenase module to the respiratory chain. So far, it was not possible to isolate the MBH in its native heterotrimeric state due to the loss of HoxZ during the process of protein solubilization. By using the very mild detergent digitonin, we were successful in isolating the MBH hydrogenase module in complex with the cytochrome b. H(2)-dependent reduction of the two HoxZ-stemming heme centers demonstrated that the hydrogenase module is productively connected to the cytochrome b. Further investigation provided evidence that the MBH exists in the membrane as a high molecular mass complex consisting of three heterotrimeric units. The lipids phosphatidylethanolamine and phosphatidylglycerol were identified to play a role in the interaction of the hydrogenase module with the cytochrome b subunit.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Bacterial Outer Membrane Proteins / chemistry
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Bacterial Outer Membrane Proteins / genetics
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Bacterial Outer Membrane Proteins / isolation & purification*
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Bacterial Outer Membrane Proteins / metabolism
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Cardiolipins / metabolism
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Cupriavidus necator / enzymology*
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Cupriavidus necator / metabolism
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Cytochrome b Group / chemistry
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Cytochrome b Group / genetics
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Cytochrome b Group / isolation & purification*
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Cytochrome b Group / metabolism
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Digitonin / chemistry
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Enzyme Stability
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Hydrogenase / chemistry
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Hydrogenase / genetics
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Hydrogenase / isolation & purification*
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Hydrogenase / metabolism
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Models, Molecular
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Molecular Weight
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Multiprotein Complexes / chemistry
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Multiprotein Complexes / genetics
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Multiprotein Complexes / isolation & purification
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Multiprotein Complexes / metabolism
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Oxidation-Reduction
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Phosphatidylethanolamines / metabolism
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Phosphatidylglycerols / metabolism
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Protein Multimerization
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Protein Subunits / chemistry
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Protein Subunits / genetics
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Protein Subunits / isolation & purification*
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Protein Subunits / metabolism
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / isolation & purification
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Recombinant Fusion Proteins / metabolism
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Surface-Active Agents / chemistry
Substances
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Bacterial Outer Membrane Proteins
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Cardiolipins
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Cytochrome b Group
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Multiprotein Complexes
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Phosphatidylethanolamines
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Phosphatidylglycerols
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Protein Subunits
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Recombinant Fusion Proteins
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Surface-Active Agents
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nickel-iron hydrogenase
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Hydrogenase
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Digitonin