Cupriavidus necator

Cupriavidus necator
Scientific classification
Domain:	Bacteria
Phylum:	Pseudomonadota
Class:	Betaproteobacteria
Order:	Burkholderiales
Family:	Burkholderiaceae
Genus:	Cupriavidus
Species:	C. necator
Binomial name
Cupriavidus necator (Davis 1969) Yabuuchi et al. 1996
Synonyms
Ralstonia eutropha

Cupriavidus necator is a

Cupriavidus necator can use hydrogen gas as a source of energy when growing under autotrophic conditions. It contains four different hydrogenases that have [Ni-Fe] active sites and all perform this reaction:^[14][15]

H₂ ${\displaystyle \rightleftharpoons }$ 2H⁺ + 2e⁻

The hydrogenases of C. necator are like other typical [Ni-Fe] hydrogenases because they are made up of a large and a small subunit. The large subunit is where the [Ni-Fe] active site resides and the small subunit is composed of [Fe-S] clusters.^[16] However, the hydrogenases of C. necator are different from typical [Ni-Fe] hydrogenases because they are tolerant to oxygen and are not inhibited by CO.^[14] While the four hydrogenases perform the same reaction in the cell, each hydrogenase is linked to a different cellular process. The differences between the regulatory hydrogenase, membrane-bound hydrogenase, soluble hydrogenase and actinobacterial hydrogenase in C. necator are described below.

Regulatory hydrogenase

The first hydrogenase is a regulatory hydrogenase (RH) that signals to the cell hydrogen is present. The RH is a protein containing large and small [Ni-Fe] hydrogenase subunits attached to a histidine protein kinase subunit.^[17] The hydrogen gas is oxidized at the [Ni-Fe] center in the large subunit and in turn reduces the [Fe-S] clusters in the small subunit. It is unknown whether the electrons are transferred from the [Fe-S] clusters to the protein kinase domain.^[14] The histidine protein kinase activates a response regulator. The response regulator is active in the dephosphorylated form. The dephosphorylated response regulator promotes the transcription of the membrane bound hydrogenase and soluble hydrogenase.^[18]

Membrane-bound hydrogenase

The membrane-bound hydrogenase (MBH) is linked to the respiratory chain through a specific cytochrome b-related protein in C. necator.^[19] Hydrogen gas is oxidized at the [Ni-Fe] active site in the large subunit and the electrons are shuttled through the [Fe-S] clusters in the small subunit to the cytochrome b-like protein.^[14] The MBH is located on the outer cytoplasmic membrane. It recovers energy for the cell by funneling electrons into the respiratory chain and by increasing the proton gradient.^[19] The MBH in C. necator is not inhibited by CO and is tolerant to oxygen.^[20]

NAD+-reducing hydrogenase

The NAD+-reducing hydrogenase (soluble hydrogenase, SH) creates a NADH-reducing equivalence by oxidizing hydrogen gas. The SH is a heterohexameric protein^[21] with two subunits making up the large and small subunits of the [Ni-Fe] hydrogenase and the other two subunits comprising a reductase module similar to the one of Complex I.^[22] The [Ni-Fe] active site oxidized hydrogen gas which transfers electrons to a FMN-a cofactor, then to a [Fe-S] cluster relay of the small hydrogenase subunit and the reductase module, then to another FMN-b cofactor and finally to NAD⁺.^[14] The reducing equivalences are then used for fixing carbon dioxide when C. necator is growing autotrophically.

The active site of the SH of C. necator H16 has been extensively studied because C. necator H16 can be produced in large amounts, can be genetically manipulated, and can be analyzed with spectrographic techniques. However, no crystal structure is currently available for the C. necator H16 soluble hydrogenase in the presence of oxygen to determine the interactions of the active site with the rest of the protein.^[14]

Typical anaerobic [Ni-Fe] hydrogenases

The [Ni-Fe] hydrogenase from

The SH in C. necator are unique for other organisms because it is oxygen tolerant.[27] The active site of the SH has been studied to learn why this protein is tolerant to oxygen. A recent study showed that oxygen tolerance as implemented in the SH is based on a continuous catalytically driven detoxification of O₂ [Ref missing].  The genes encoding this SH can be up-regulated under heterotrophic growth condition using glycerol in the growth media ^[28] and this enables aerobic production and purification of the same enzyme.^[29]

Applications

The oxygen-tolerant hydrogenases of C. necator have been studied for diverse purposes. C. necator was studied as an attractive organism to help support life in space. It can fix carbon dioxide as a carbon source, use the urea in urine as a nitrogen source, and use hydrogen as an energy source to create dense cultures that could be used as a source of protein.^[30][31]

Electrolysis of water is one way of creating oxygenic atmosphere in space and C. necator was investigated to recycle the hydrogen produced during this process.^[32]

Oxygen-tolerant hydrogenases are being used to investigate biofuels. Hydrogenases from C. necator have been used to coat electrode surfaces to create hydrogen fuel cells tolerant to oxygen and carbon monoxide^[20] and to design hydrogen-producing light complexes.^[33] In addition, the hydrogenases from C. necator have been used to create hydrogen sensors.^[34] Genetically modified C. necator can produce isobutanol from CO
₂ that can directly substitute or blend with gasoline. The organism emits the isobutanol without having to be destroyed to obtain it.^[35]

Industrial uses

Researchers at UCLA have genetically modified a strain of the species C. necator (formerly known as R. eutropha H16) to produce isobutanol from CO₂ feedstock using electricity produced by a solar cell. The project, funded by the U.S. Dept. of Energy, is a potential high energy-density electrofuel that could use existing infrastructure to replace oil as a transportation fuel.^[36]

Chemical and biomolecular engineers at