Probing the Origin of the Metabolic Precursor of the CO Ligand in the Catalytic Center of [NiFe] Hydrogenase

Bürstel, Ingmar; Hummel, Philipp; Siebert, Elisabeth; Wisitruangsakul, Nattawadee; Zebger, Ingo; Friedrich, Bärbel; Lenz, Oliver

doi:10.1074/jbc.m111.309351

Cited by 32 publications

(38 citation statements)

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“…S1). This supports the assumption of two independent pathways for CO ligand synthesis (11). At low cell density, correlating with exposure to high O 2 tension, HypX turns out to be crucial for biosynthesis of the carbonyl ligand.…”

Section: Resultssupporting

confidence: 66%

“…Heterotrophic growth of R. eutropha with 13 C-glycerol as the sole source of carbon and energy led to a fully labeled CO ligand in hydrogenase, demonstrating that the carbonyl moiety originates from the cellular metabolism. Remarkably, selective removal of CO gas, which was released by R. eutropha cells during lithoautotrophic growth on H 2 and CO 2 in the presence of high O 2 concentrations, caused a considerable growth delay due to a reduced amount of fully maturated hydrogenase (11). Interestingly, a similar growth retardation was observed for a R. eutropha mutant deleted for the hypX gene (12).…”

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confidence: 50%

“…Maturation studies on the O 2 -tolerant, energy-generating [NiFe]-hydrogenases in the facultative H 2 -oxidizing bacterium Ralstonia eutropha H16 indicate that at least two different metabolic sources exist for CO ligand synthesis (11). Heterotrophic growth of R. eutropha with 13 C-glycerol as the sole source of carbon and energy led to a fully labeled CO ligand in hydrogenase, demonstrating that the carbonyl moiety originates from the cellular metabolism.…”

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confidence: 99%

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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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confidence: 66%

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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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“…The cyano group is transferred to the HypCD complex (8), which is thought to catalyze the formation of the Fe(CN) 2 CO group and insert the Fe ligand into the hydrogenase large subunit (9)(10)(11)(12). The biological origin of the CO molecule is still unclear (13). After the Fe atom insertion, HypA and HypB insert the Ni atom into the hydrogenase large subunit (14).…”

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confidence: 99%

Crystal structures of the carbamoylated and cyanated forms of HypE for [NiFe] hydrogenase maturation

Tominaga

Watanabe

Matsumi

et al. 2013

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

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Hydrogenase pleiotropically acting protein (Hyp)E plays a role in biosynthesis of the cyano groups for the NiFe(CN) 2 CO center of [NiFe] hydrogenases by catalyzing the ATP-dependent dehydration of the carbamoylated C-terminal cysteine of HypE to thiocyanate. Although structures of HypE proteins have been determined, until now there has been no structural evidence to explain how HypE dehydrates thiocarboxamide into thiocyanate. Here, we report the crystal structures of the carbamoylated and cyanated forms of HypE from Thermococcus kodakarensis in complex with nucleotides at 1.53-and 1.64-Å resolution, respectively. Carbamoylation of the C-terminal cysteine (Cys338) of HypE by chemical modification is clearly observed in the present structures. In the presence of ATP, the thiocarboxamide of Cys338 is successfully dehydrated into the thiocyanate. In the carbamoylated state, the thiocarboxamide nitrogen atom of Cys338 is close to a conserved glutamate residue (Glu272), but the spatial position of Glu272 is less favorable for proton abstraction. On the other hand, the thiocarboxamide oxygen atom of Cys338 interacts with a conserved lysine residue (Lys134) through a water molecule. The close contact of Lys134 with an arginine residue lowers the pK a of Lys134, suggesting that Lys134 functions as a proton acceptor. These observations suggest that the dehydration of thiocarboxamide into thiocyanate is catalyzed by a two-step deprotonation process, in which Lys134 and Glu272 function as the first and second bases, respectively.

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“…The source and synthetic pathway of the CO ligand remain unknown, although the involvement of HypD has been proposed (8). A recent isotope labeling experiment has suggested that the extrinsic CO molecule may be incorporated into the bimetallic cluster but there seems to be a metabolic pathway for the synthesis of the carbonyl ligand (16).…”

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Structural Basis for the Reaction Mechanism of S-Carbamoylation of HypE by HypF in the Maturation of [NiFe]-Hydrogenases

Shomura

Higuchi

2012

Journal of Biological Chemistry

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Probing the Origin of the Metabolic Precursor of the CO Ligand in the Catalytic Center of [NiFe] Hydrogenase

Cited by 32 publications

References 45 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

Crystal structures of the carbamoylated and cyanated forms of HypE for [NiFe] hydrogenase maturation

Structural Basis for the Reaction Mechanism of S-Carbamoylation of HypE by HypF in the Maturation of [NiFe]-Hydrogenases

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Probing the Origin of the Metabolic Precursor of the CO Ligand in the Catalytic Center of [NiFe] Hydrogenase

Cited by 32 publications

References 45 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

Crystal structures of the carbamoylated and cyanated forms of HypE for [NiFe] hydrogenase maturation

Structural Basis for the Reaction Mechanism of S-Carbamoylation of HypE by HypF in the Maturation of [NiFe]-Hydrogenases

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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