4 Oxygen-tolerant hydrogenases and their biotechnological potential

Lenz, Oliver; Lauterbach, Lars; Frielingsdorf, Stefan; Friedrich, Bärbel

doi:10.1515/9783110336733.61

Cited by 28 publications

(17 citation statements)

References 111 publications

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“…Of the four hydrogenases present in R. eutropha (18), we selected the regulatory [NiFe]-hydrogenase (RH) as the model system for our study. The RH undergoes a less complex maturation process than the other three hydrogenases (18); it can be conveniently purified from soluble cell extracts by affinity chromatography, and the carbonyl and cyanide ligands of the active site are easily accessible to IR spectroscopic analysis (11). An almost quantitative labeling of the carbonyl ligand was observed in the IR spectrum of the RH isolated from cells of the HypX + strain (Fig.…”

Section: Resultsmentioning

confidence: 99%

CO synthesized from the central one-carbon pool as source for the iron carbonyl in O ₂ -tolerant [NiFe]-hydrogenase

Bürstel

Siebert

Frielingsdorf

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Hydrogenases are nature's key catalysts involved in both microbial consumption and production of molecular hydrogen. H 2 exhibits a strongly bonded, almost inert electron pair and requires transition metals for activation. Consequently, all hydrogenases are metalloenzymes that contain at least one iron atom in the catalytic center. For appropriate interaction with H 2 , the iron moiety demands for a sophisticated coordination environment that cannot be provided just by standard amino acids. This dilemma has been overcome by the introduction of unprecedented chemistry-that is, by ligating the iron with carbon monoxide (CO) and cyanide (or equivalent) groups. These ligands are both unprecedented in microbial metabolism and, in their free form, highly toxic to living organisms. Therefore, the formation of the diatomic ligands relies on dedicated biosynthesis pathways. So far, biosynthesis of the CO ligand in [NiFe]-hydrogenases was unknown. Here we show that the aerobic H 2 oxidizer Ralstonia eutropha, which produces active [NiFe]-hydrogenases in the presence of O 2 , employs the auxiliary protein HypX (hydrogenase pleiotropic maturation X) for CO ligand formation. Using genetic engineering and isotope labeling experiments in combination with infrared spectroscopic investigations, we demonstrate that the α-carbon of glycine ends up in the CO ligand of [NiFe]-hydrogenase. The α-carbon of glycine is a building block of the central one-carbon metabolism intermediate, N 10 -formyl-tetrahydrofolate (N 10 -CHO-THF). Evidence is presented that the multidomain protein, HypX, converts the formyl group of N 10 -CHO-THF into water and CO, thereby providing the carbonyl ligand for hydrogenase. This study contributes insights into microbial biosynthesis of metal carbonyls involving toxic intermediates.H ydrogenases are abundant metalloenzymes in prokaryotes and lower eukaryotes in which they catalyze the reversible oxidation of molecular hydrogen into protons and electrons. Depending on the physiological conditions, hydrogenases enable their hosts either to use hydrogen as an energy source or to dissipate excess, reducing power as molecular hydrogen (1, 2). Enzymatic cycling of H 2 is characterized by high substrate specificity and high turnover rates and has received great attention from both fundamental and applied perspectives (3).The two major classes of hydrogenases, [FeFe]-and [NiFe]-hydrogenases, are grouped on the basis of their metal content in the catalytic center. Although their active site structures differ considerably, the two hydrogenase types share uncommon, nonproteinaceous diatomic iron ligands. The diiron site of [FeFe]-hydrogenases is equipped with two cyanide (CN − ) and three carbon monoxide (CO) molecules, whereas the active site iron of [NiFe]-hydrogenases ligates two CN − residues and one CO (1-5). Biosynthesis of these diatomic ligands involves intriguing chemistry, which is challenging for a living cell because of the toxicity of free CN − and CO molecules. In the case of [NiFe]-hydrogenases, at least...

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Section: Resultsmentioning

confidence: 99%

CO synthesized from the central one-carbon pool as source for the iron carbonyl in O ₂ -tolerant [NiFe]-hydrogenase

Bürstel

Siebert

Frielingsdorf

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…This is in-line with the low H 2 turnover rate observed for H 2 sensors, which, in turn, prevents energy loss and saves H 2 for the energy-converting hydrogenases. 36 …”

Section: Discussionmentioning

confidence: 99%

“… 32 – 34 The absence of the Ni-A and Ni-B states in RH, both of which require reductive reactivation, is fully consistent with the sensory character of the protein that needs to react immediately in the presence of H 2 . 22 , 24 , 35 , 36 Incubation of RH with H 2 in absence of artificial electron acceptors affords the paramagnetic Ni-C state characterized by a hydride ligand in the bridging position between the Ni III and Fe II ions. 20 This state appears to be a thermodynamic “bottleneck”.…”

Section: Introductionmentioning

confidence: 99%

Cofactor composition and function of a H₂-sensing regulatory hydrogenase as revealed by Mössbauer and EPR spectroscopy

et al. 2015

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“…6 Synthesis of these enzymes is controlled at the transcriptional level by a H 2 -sensing system involving a regulatory Ni-Fe hydrogenase (RH), a histidine protein kinase, and a response regulator. 9 The regulatory hydrogenase is composed of the subunits HoxBC and occurs natively as a pair of HoxBC units that is associated with a histidine protein kinase tetramer. 10,11 Nickel−iron hydrogenases have differing levels of sensitivity to dioxygen.…”

Section: ■ Introductionmentioning

confidence: 99%

Electrochemical and Infrared Spectroscopic Studies Provide Insight into Reactions of the NiFe Regulatory Hydrogenase from Ralstonia eutropha with O₂ and CO

et al. 2015

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The regulatory hydrogenase (RH) from Ralstonia eutropha acts as the H2-sensing unit of a two-component system that regulates biosynthesis of the energy conserving hydrogenases of the organism according to the availability of H2. The H2 oxidation activity, which was so far determined in vitro with artificial electron acceptors, has been considered to be insensitive to O2 and CO. It is assumed that bulky isoleucine and phenylalanine amino acid residues close to the NiFe active site "gate" gas access, preventing molecules larger than H2 interacting with the active site. We have carried out sensitive electrochemical measurements to demonstrate that O2 is in fact an inhibitor of H2 oxidation by the RH, and that both H(+) reduction and H2 oxidation are inhibited by CO. Furthermore, we have demonstrated that the inhibitory effect of O2 arises due to interaction of O2 with the active site. Using protein film infrared electrochemistry (PFIRE) under H2 oxidation conditions, in conjunction with solution infrared measurements, we have identified previously unreported oxidized inactive and catalytically active reduced states of the RH active site. These findings suggest that the RH has a rich active site chemistry similar to that of other NiFe hydrogenases.

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4 Oxygen-tolerant hydrogenases and their biotechnological potential

Cited by 28 publications

References 111 publications

CO synthesized from the central one-carbon pool as source for the iron carbonyl in O ₂ -tolerant [NiFe]-hydrogenase

CO synthesized from the central one-carbon pool as source for the iron carbonyl in O ₂ -tolerant [NiFe]-hydrogenase

Cofactor composition and function of a H₂-sensing regulatory hydrogenase as revealed by Mössbauer and EPR spectroscopy

Electrochemical and Infrared Spectroscopic Studies Provide Insight into Reactions of the NiFe Regulatory Hydrogenase from Ralstonia eutropha with O₂ and CO

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4 Oxygen-tolerant hydrogenases and their biotechnological potential

Cited by 28 publications

References 111 publications

CO synthesized from the central one-carbon pool as source for the iron carbonyl in O 2 -tolerant [NiFe]-hydrogenase

CO synthesized from the central one-carbon pool as source for the iron carbonyl in O 2 -tolerant [NiFe]-hydrogenase

Cofactor composition and function of a H2-sensing regulatory hydrogenase as revealed by Mössbauer and EPR spectroscopy

Electrochemical and Infrared Spectroscopic Studies Provide Insight into Reactions of the NiFe Regulatory Hydrogenase from Ralstonia eutropha with O2 and CO

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CO synthesized from the central one-carbon pool as source for the iron carbonyl in O ₂ -tolerant [NiFe]-hydrogenase

CO synthesized from the central one-carbon pool as source for the iron carbonyl in O ₂ -tolerant [NiFe]-hydrogenase

Cofactor composition and function of a H₂-sensing regulatory hydrogenase as revealed by Mössbauer and EPR spectroscopy

Electrochemical and Infrared Spectroscopic Studies Provide Insight into Reactions of the NiFe Regulatory Hydrogenase from Ralstonia eutropha with O₂ and CO