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Cell-free H-cluster synthesis and [FeFe] hydrogenase activation: all five CO and CN⁻ ligands derive from tyrosine.

Kuchenreuther JM, George SJ, Grady-Smith CS, Cramer SP, Swartz JR - PLoS ONE (2011)

Bottom Line: In this report, we describe effective cell-free methods for investigating H-cluster synthesis and [FeFe] hydrogenase activation.Combining isotopic labeling with FTIR spectroscopy, we conclusively show that each of the CO and CN⁻ ligands derive respectively from the carboxylate and amino substituents of tyrosine.Such in vitro systems with reconstituted pathways comprise a versatile approach for studying biosynthetic mechanisms, and this work marks a significant step towards an understanding of both the protein-protein interactions and complex reactions required for H-cluster assembly and hydrogenase maturation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Stanford University, Stanford, California, United States of America.

ABSTRACT
[FeFe] hydrogenases are promising catalysts for producing hydrogen as a sustainable fuel and chemical feedstock, and they also serve as paradigms for biomimetic hydrogen-evolving compounds. Hydrogen formation is catalyzed by the H-cluster, a unique iron-based cofactor requiring three carbon monoxide (CO) and two cyanide (CN⁻) ligands as well as a dithiolate bridge. Three accessory proteins (HydE, HydF, and HydG) are presumably responsible for assembling and installing the H-cluster, yet their precise roles and the biosynthetic pathway have yet to be fully defined. In this report, we describe effective cell-free methods for investigating H-cluster synthesis and [FeFe] hydrogenase activation. Combining isotopic labeling with FTIR spectroscopy, we conclusively show that each of the CO and CN⁻ ligands derive respectively from the carboxylate and amino substituents of tyrosine. Such in vitro systems with reconstituted pathways comprise a versatile approach for studying biosynthetic mechanisms, and this work marks a significant step towards an understanding of both the protein-protein interactions and complex reactions required for H-cluster assembly and hydrogenase maturation.

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In vitro [FeFe] hydrogenase activation for FTIR spectroscopic analysis.(Fig. 1A) A ball and stick representation of the hydrogenase H-cluster. The catalytic [2Fe]H cluster is joined to the cubane [4Fe]H cluster, colored with the following scheme: brown (Fe), yellow (S), gray (C), red (O), blue (N), and magenta (unknown). (Fig. 1B) The chemical structure for L-tyrosine, with carbon atoms numbered 1–9. (Fig. 1C) The in vitro hydrogenase maturation process. For cell-free H-cluster synthesis, (1) CpI apoenzyme (PDB ID 3C8Y) as well as (2) exogenous substrates are added to (3) a mixture of three lysates containing E. coli proteins (yellow ovals) and individually produced maturases. HydE, HydF, and HydG are expressed separately to avoid H-cluster synthesis during in vivo maturase expression. Following hydrogenase maturation, (4) the CpI holoenzyme is re-purified, and (5) the active hydrogenase is examined using FTIR spectroscopy.
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pone-0020346-g001: In vitro [FeFe] hydrogenase activation for FTIR spectroscopic analysis.(Fig. 1A) A ball and stick representation of the hydrogenase H-cluster. The catalytic [2Fe]H cluster is joined to the cubane [4Fe]H cluster, colored with the following scheme: brown (Fe), yellow (S), gray (C), red (O), blue (N), and magenta (unknown). (Fig. 1B) The chemical structure for L-tyrosine, with carbon atoms numbered 1–9. (Fig. 1C) The in vitro hydrogenase maturation process. For cell-free H-cluster synthesis, (1) CpI apoenzyme (PDB ID 3C8Y) as well as (2) exogenous substrates are added to (3) a mixture of three lysates containing E. coli proteins (yellow ovals) and individually produced maturases. HydE, HydF, and HydG are expressed separately to avoid H-cluster synthesis during in vivo maturase expression. Following hydrogenase maturation, (4) the CpI holoenzyme is re-purified, and (5) the active hydrogenase is examined using FTIR spectroscopy.

Mentions: Hydrogenases contain complex [FeFe]-, [NiFe]-, or [Fe]-based catalytic cofactors that are stabilized by multiple non-protein ligands [8]. [FeFe] hydrogenases are the fastest H2 producers and require the H-cluster, a catalytic cofactor comprised of two iron-based clusters connected via a cysteinyl sulfur atom (Fig. 1). The cubane Fe–S cluster ([4Fe]H) presumably delivers electrons to the catalytic 2Fe unit ([2Fe]H), which contains three carbon monoxide (CO) and two cyanide (CN−) adducts as well as a dithiol bridging group of disputed composition [9], [10]. Three proteins called the HydE, HydF, and HydG maturases participate in the synthesis of the H-cluster and the activation of [FeFe] hydrogenases [11]. The final maturation step presumably occurs when the HydF maturase transfers the [2Fe]H cluster to the hydrogenase [12], [13], likely through a positively charged channel as proposed by Mulder et al. [14].


Cell-free H-cluster synthesis and [FeFe] hydrogenase activation: all five CO and CN⁻ ligands derive from tyrosine.

Kuchenreuther JM, George SJ, Grady-Smith CS, Cramer SP, Swartz JR - PLoS ONE (2011)

In vitro [FeFe] hydrogenase activation for FTIR spectroscopic analysis.(Fig. 1A) A ball and stick representation of the hydrogenase H-cluster. The catalytic [2Fe]H cluster is joined to the cubane [4Fe]H cluster, colored with the following scheme: brown (Fe), yellow (S), gray (C), red (O), blue (N), and magenta (unknown). (Fig. 1B) The chemical structure for L-tyrosine, with carbon atoms numbered 1–9. (Fig. 1C) The in vitro hydrogenase maturation process. For cell-free H-cluster synthesis, (1) CpI apoenzyme (PDB ID 3C8Y) as well as (2) exogenous substrates are added to (3) a mixture of three lysates containing E. coli proteins (yellow ovals) and individually produced maturases. HydE, HydF, and HydG are expressed separately to avoid H-cluster synthesis during in vivo maturase expression. Following hydrogenase maturation, (4) the CpI holoenzyme is re-purified, and (5) the active hydrogenase is examined using FTIR spectroscopy.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105041&req=5

pone-0020346-g001: In vitro [FeFe] hydrogenase activation for FTIR spectroscopic analysis.(Fig. 1A) A ball and stick representation of the hydrogenase H-cluster. The catalytic [2Fe]H cluster is joined to the cubane [4Fe]H cluster, colored with the following scheme: brown (Fe), yellow (S), gray (C), red (O), blue (N), and magenta (unknown). (Fig. 1B) The chemical structure for L-tyrosine, with carbon atoms numbered 1–9. (Fig. 1C) The in vitro hydrogenase maturation process. For cell-free H-cluster synthesis, (1) CpI apoenzyme (PDB ID 3C8Y) as well as (2) exogenous substrates are added to (3) a mixture of three lysates containing E. coli proteins (yellow ovals) and individually produced maturases. HydE, HydF, and HydG are expressed separately to avoid H-cluster synthesis during in vivo maturase expression. Following hydrogenase maturation, (4) the CpI holoenzyme is re-purified, and (5) the active hydrogenase is examined using FTIR spectroscopy.
Mentions: Hydrogenases contain complex [FeFe]-, [NiFe]-, or [Fe]-based catalytic cofactors that are stabilized by multiple non-protein ligands [8]. [FeFe] hydrogenases are the fastest H2 producers and require the H-cluster, a catalytic cofactor comprised of two iron-based clusters connected via a cysteinyl sulfur atom (Fig. 1). The cubane Fe–S cluster ([4Fe]H) presumably delivers electrons to the catalytic 2Fe unit ([2Fe]H), which contains three carbon monoxide (CO) and two cyanide (CN−) adducts as well as a dithiol bridging group of disputed composition [9], [10]. Three proteins called the HydE, HydF, and HydG maturases participate in the synthesis of the H-cluster and the activation of [FeFe] hydrogenases [11]. The final maturation step presumably occurs when the HydF maturase transfers the [2Fe]H cluster to the hydrogenase [12], [13], likely through a positively charged channel as proposed by Mulder et al. [14].

Bottom Line: In this report, we describe effective cell-free methods for investigating H-cluster synthesis and [FeFe] hydrogenase activation.Combining isotopic labeling with FTIR spectroscopy, we conclusively show that each of the CO and CN⁻ ligands derive respectively from the carboxylate and amino substituents of tyrosine.Such in vitro systems with reconstituted pathways comprise a versatile approach for studying biosynthetic mechanisms, and this work marks a significant step towards an understanding of both the protein-protein interactions and complex reactions required for H-cluster assembly and hydrogenase maturation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Stanford University, Stanford, California, United States of America.

ABSTRACT
[FeFe] hydrogenases are promising catalysts for producing hydrogen as a sustainable fuel and chemical feedstock, and they also serve as paradigms for biomimetic hydrogen-evolving compounds. Hydrogen formation is catalyzed by the H-cluster, a unique iron-based cofactor requiring three carbon monoxide (CO) and two cyanide (CN⁻) ligands as well as a dithiolate bridge. Three accessory proteins (HydE, HydF, and HydG) are presumably responsible for assembling and installing the H-cluster, yet their precise roles and the biosynthetic pathway have yet to be fully defined. In this report, we describe effective cell-free methods for investigating H-cluster synthesis and [FeFe] hydrogenase activation. Combining isotopic labeling with FTIR spectroscopy, we conclusively show that each of the CO and CN⁻ ligands derive respectively from the carboxylate and amino substituents of tyrosine. Such in vitro systems with reconstituted pathways comprise a versatile approach for studying biosynthetic mechanisms, and this work marks a significant step towards an understanding of both the protein-protein interactions and complex reactions required for H-cluster assembly and hydrogenase maturation.

Show MeSH