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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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SDS-PAGE and Coomassie staining of purified CpI apoenzyme and E. coli lysates with heterologous maturases.All proteins were identified using the Mark12™ protein ladder (Invitrogen), and the 36, 55, and 66 kD protein standards are indicated. The control lysate from E. coli strain BL21(DE3) ΔiscR (lane 1) has no proteins produced from recombinant DNA plasmids. Maturase lysates with soluble HydE (40 kD), HydF (45 kD), or HydG (54 kD) are shown in lanes 2–4, respectively. We estimated that the cell lysates (0.25 µL of lysate loaded per lane) contained 3–15 mg·mL−1 of each maturase, and approximately 2.5 µg of CpI–Strep-tag II apoenzyme (64 kD) is shown in lane 5.
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pone-0020346-g002: SDS-PAGE and Coomassie staining of purified CpI apoenzyme and E. coli lysates with heterologous maturases.All proteins were identified using the Mark12™ protein ladder (Invitrogen), and the 36, 55, and 66 kD protein standards are indicated. The control lysate from E. coli strain BL21(DE3) ΔiscR (lane 1) has no proteins produced from recombinant DNA plasmids. Maturase lysates with soluble HydE (40 kD), HydF (45 kD), or HydG (54 kD) are shown in lanes 2–4, respectively. We estimated that the cell lysates (0.25 µL of lysate loaded per lane) contained 3–15 mg·mL−1 of each maturase, and approximately 2.5 µg of CpI–Strep-tag II apoenzyme (64 kD) is shown in lane 5.

Mentions: The work in this report would not have been possible without scalable methods for making large quantities of active [FeFe] hydrogenases in a cell-free environment. We recently improved the in vivo expression of active hydrogenases in E. coli [26], and we extended those methods for high-yield expression of the individual maturases and CpI apoenzyme. The maturase lysates used for in vitro hydrogenase maturation (Fig. 1) therefore contained high concentrations of HydE, HydF, or HydG, which we estimated to be 3–15 mg·mL−1 (Fig. 2). This was crucial to achieve nearly complete activation of the CpI hydrogenase (Table 1) at concentrations of ∼200 mg·L−1, more than 300-fold higher than with methods that lack in vitro H-cluster synthesis [12], [27]. By using non-purified maturation proteins, the activation reaction volumes could be increased to more than 100 mL, which allowed us to produce and re-purify the milligram quantities of CpI hydrogenase required for spectroscopic analysis.


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)

SDS-PAGE and Coomassie staining of purified CpI apoenzyme and E. coli lysates with heterologous maturases.All proteins were identified using the Mark12™ protein ladder (Invitrogen), and the 36, 55, and 66 kD protein standards are indicated. The control lysate from E. coli strain BL21(DE3) ΔiscR (lane 1) has no proteins produced from recombinant DNA plasmids. Maturase lysates with soluble HydE (40 kD), HydF (45 kD), or HydG (54 kD) are shown in lanes 2–4, respectively. We estimated that the cell lysates (0.25 µL of lysate loaded per lane) contained 3–15 mg·mL−1 of each maturase, and approximately 2.5 µg of CpI–Strep-tag II apoenzyme (64 kD) is shown in lane 5.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105041&req=5

pone-0020346-g002: SDS-PAGE and Coomassie staining of purified CpI apoenzyme and E. coli lysates with heterologous maturases.All proteins were identified using the Mark12™ protein ladder (Invitrogen), and the 36, 55, and 66 kD protein standards are indicated. The control lysate from E. coli strain BL21(DE3) ΔiscR (lane 1) has no proteins produced from recombinant DNA plasmids. Maturase lysates with soluble HydE (40 kD), HydF (45 kD), or HydG (54 kD) are shown in lanes 2–4, respectively. We estimated that the cell lysates (0.25 µL of lysate loaded per lane) contained 3–15 mg·mL−1 of each maturase, and approximately 2.5 µg of CpI–Strep-tag II apoenzyme (64 kD) is shown in lane 5.
Mentions: The work in this report would not have been possible without scalable methods for making large quantities of active [FeFe] hydrogenases in a cell-free environment. We recently improved the in vivo expression of active hydrogenases in E. coli [26], and we extended those methods for high-yield expression of the individual maturases and CpI apoenzyme. The maturase lysates used for in vitro hydrogenase maturation (Fig. 1) therefore contained high concentrations of HydE, HydF, or HydG, which we estimated to be 3–15 mg·mL−1 (Fig. 2). This was crucial to achieve nearly complete activation of the CpI hydrogenase (Table 1) at concentrations of ∼200 mg·L−1, more than 300-fold higher than with methods that lack in vitro H-cluster synthesis [12], [27]. By using non-purified maturation proteins, the activation reaction volumes could be increased to more than 100 mL, which allowed us to produce and re-purify the milligram quantities of CpI hydrogenase required for spectroscopic analysis.

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
Related in: MedlinePlus