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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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FTIR spectroscopic analysis for active CpI produced in vitro using natural abundance tyrosine or isotopically labeled tyrosine analogs.The IR spectra are for the as-isolated active CpI hydrogenase containing the H-cluster produced in the presence of L-tyrosine (CpItyr), L-[2-13C]-tyrosine (CpI2-13C-tyr), L-[1-13C]-tyrosine (CpI1-13C-tyr), and L-[U-13C-15N]-tyrosine (CpIU-13C-15N-tyr). The shifts in vibrational energies correlate with expected changes for ν(13CO), ν(13CN), and ν(13C15N) modes, confirming that the CO and CN− ligands are synthesized from tyrosine. Labels indicating the assigned ν(CO) and ν(CN) vibrational modes are provided at the top of the figure, with the 13CN−/13C15N− and 13CO ligands shown in red and green, respectively, in the molecular diagrams. Vertical scale bars provided at 1740 cm−1 represent a difference of 0.5 milliabsorbance units. Table 2 summarizes the vibrational energies and corresponding assigned ν(CN) and ν(CO) modes for the Hox clusters.
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pone-0020346-g003: FTIR spectroscopic analysis for active CpI produced in vitro using natural abundance tyrosine or isotopically labeled tyrosine analogs.The IR spectra are for the as-isolated active CpI hydrogenase containing the H-cluster produced in the presence of L-tyrosine (CpItyr), L-[2-13C]-tyrosine (CpI2-13C-tyr), L-[1-13C]-tyrosine (CpI1-13C-tyr), and L-[U-13C-15N]-tyrosine (CpIU-13C-15N-tyr). The shifts in vibrational energies correlate with expected changes for ν(13CO), ν(13CN), and ν(13C15N) modes, confirming that the CO and CN− ligands are synthesized from tyrosine. Labels indicating the assigned ν(CO) and ν(CN) vibrational modes are provided at the top of the figure, with the 13CN−/13C15N− and 13CO ligands shown in red and green, respectively, in the molecular diagrams. Vertical scale bars provided at 1740 cm−1 represent a difference of 0.5 milliabsorbance units. Table 2 summarizes the vibrational energies and corresponding assigned ν(CN) and ν(CO) modes for the Hox clusters.

Mentions: The vibrational energies and corresponding n(CN) and n(CO) mode assignments are provided for each Hox cluster from active CpI produced with either unlabeled or isotopically labeled tyrosine. Energies were determined from spectra measured using FTIR spectroscopy (Fig. 3). The spectrum for each isotopically labeled sample also contains low intensity bands indicating trace amounts of unlabeled CO and CN−incorporated into the H-cluster. The intensities of these bands vary from sample to sample, and they do not depend on the location of either CO or CN− on the H-cluster. We thus attribute these features to either adventitious free tyrosine present in the cell lysates or possibly to low quantities of an iron cluster with CO and CN− ligands that is pre-assembled by a single Hyd maturase during in vivo expression. Each spectrum also shows evidence for CpI with reduced H-cluster (Hred), characterized in the CpItyr case by bands located at 2053 cm−1, 2039 cm−1, 1961 cm−1, 1914 cm−1, and 1899 cm−1.


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)

FTIR spectroscopic analysis for active CpI produced in vitro using natural abundance tyrosine or isotopically labeled tyrosine analogs.The IR spectra are for the as-isolated active CpI hydrogenase containing the H-cluster produced in the presence of L-tyrosine (CpItyr), L-[2-13C]-tyrosine (CpI2-13C-tyr), L-[1-13C]-tyrosine (CpI1-13C-tyr), and L-[U-13C-15N]-tyrosine (CpIU-13C-15N-tyr). The shifts in vibrational energies correlate with expected changes for ν(13CO), ν(13CN), and ν(13C15N) modes, confirming that the CO and CN− ligands are synthesized from tyrosine. Labels indicating the assigned ν(CO) and ν(CN) vibrational modes are provided at the top of the figure, with the 13CN−/13C15N− and 13CO ligands shown in red and green, respectively, in the molecular diagrams. Vertical scale bars provided at 1740 cm−1 represent a difference of 0.5 milliabsorbance units. Table 2 summarizes the vibrational energies and corresponding assigned ν(CN) and ν(CO) modes for the Hox clusters.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020346-g003: FTIR spectroscopic analysis for active CpI produced in vitro using natural abundance tyrosine or isotopically labeled tyrosine analogs.The IR spectra are for the as-isolated active CpI hydrogenase containing the H-cluster produced in the presence of L-tyrosine (CpItyr), L-[2-13C]-tyrosine (CpI2-13C-tyr), L-[1-13C]-tyrosine (CpI1-13C-tyr), and L-[U-13C-15N]-tyrosine (CpIU-13C-15N-tyr). The shifts in vibrational energies correlate with expected changes for ν(13CO), ν(13CN), and ν(13C15N) modes, confirming that the CO and CN− ligands are synthesized from tyrosine. Labels indicating the assigned ν(CO) and ν(CN) vibrational modes are provided at the top of the figure, with the 13CN−/13C15N− and 13CO ligands shown in red and green, respectively, in the molecular diagrams. Vertical scale bars provided at 1740 cm−1 represent a difference of 0.5 milliabsorbance units. Table 2 summarizes the vibrational energies and corresponding assigned ν(CN) and ν(CO) modes for the Hox clusters.
Mentions: The vibrational energies and corresponding n(CN) and n(CO) mode assignments are provided for each Hox cluster from active CpI produced with either unlabeled or isotopically labeled tyrosine. Energies were determined from spectra measured using FTIR spectroscopy (Fig. 3). The spectrum for each isotopically labeled sample also contains low intensity bands indicating trace amounts of unlabeled CO and CN−incorporated into the H-cluster. The intensities of these bands vary from sample to sample, and they do not depend on the location of either CO or CN− on the H-cluster. We thus attribute these features to either adventitious free tyrosine present in the cell lysates or possibly to low quantities of an iron cluster with CO and CN− ligands that is pre-assembled by a single Hyd maturase during in vivo expression. Each spectrum also shows evidence for CpI with reduced H-cluster (Hred), characterized in the CpItyr case by bands located at 2053 cm−1, 2039 cm−1, 1961 cm−1, 1914 cm−1, and 1899 cm−1.

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