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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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Infrared spectra for CpI hydrogenase with isotopically labeled H-cluster containing exogenously bound CO.The infrared spectra are for the CO-inhibited CpI enzyme harboring an 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). Natural abundance COexo was added to CpItyr and CpI2-13C-tyr, which have intrinsic CO ligands. Conversely, 13COexo was added to CpI1-13C-tyr and CpIU-13C-15N-tyr, which have intrinsic 13CO ligands. Comparing the Hox–COexo spectrum for each CpI sample to its respective Hox spectrum (Fig. 3), shifts of 5–10 cm−1 were observed for the ν(CN) modes and the ν(μ–CO) mode in all four cases. The ν(CO) mode for the Fep–CO ligand did not change. Meanwhile, the ν(CO) mode for the Fed–CO moiety was replaced with two peaks resulting from symmetric and asymmetric coupled vibrational stretches, as two CO molecules of equal mass are coordinated to the Fed atom. The peak for the ν(CO)symmetric mode is visible at 2015/1970 cm−1 for CO/13CO. The ν(CO)asymmetric mode, however, cannot be distinguished because its vibrational energy is similar to the ν(CO) mode at 1972/1928 cm−1 for the Fep–CO/Fep–13CO adducts. The changes in vibrational energies, indicated by the dashed lines, correlate with expected changes for ν(13CO), ν(13CN), and ν(13C15N) modes, again confirming that the CO and CN− ligands are synthesized from tyrosine. Labels indicating the assigned ν(CO) and ν(CN) vibrational modes are provided. The 13CN/13C15N and 13CO ligands are shown in red and green, respectively, in the molecular diagrams. Vertical scale bars shown at 1740 cm−1 represent a difference of 0.5 milliabsorbance units. Table 3 summarizes the vibrational energies and corresponding assigned ν(CN) and ν(CO) modes for the Hox–COexo clusters.
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pone-0020346-g004: Infrared spectra for CpI hydrogenase with isotopically labeled H-cluster containing exogenously bound CO.The infrared spectra are for the CO-inhibited CpI enzyme harboring an 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). Natural abundance COexo was added to CpItyr and CpI2-13C-tyr, which have intrinsic CO ligands. Conversely, 13COexo was added to CpI1-13C-tyr and CpIU-13C-15N-tyr, which have intrinsic 13CO ligands. Comparing the Hox–COexo spectrum for each CpI sample to its respective Hox spectrum (Fig. 3), shifts of 5–10 cm−1 were observed for the ν(CN) modes and the ν(μ–CO) mode in all four cases. The ν(CO) mode for the Fep–CO ligand did not change. Meanwhile, the ν(CO) mode for the Fed–CO moiety was replaced with two peaks resulting from symmetric and asymmetric coupled vibrational stretches, as two CO molecules of equal mass are coordinated to the Fed atom. The peak for the ν(CO)symmetric mode is visible at 2015/1970 cm−1 for CO/13CO. The ν(CO)asymmetric mode, however, cannot be distinguished because its vibrational energy is similar to the ν(CO) mode at 1972/1928 cm−1 for the Fep–CO/Fep–13CO adducts. The changes in vibrational energies, indicated by the dashed lines, correlate with expected changes for ν(13CO), ν(13CN), and ν(13C15N) modes, again confirming that the CO and CN− ligands are synthesized from tyrosine. Labels indicating the assigned ν(CO) and ν(CN) vibrational modes are provided. The 13CN/13C15N and 13CO ligands are shown in red and green, respectively, in the molecular diagrams. Vertical scale bars shown at 1740 cm−1 represent a difference of 0.5 milliabsorbance units. Table 3 summarizes the vibrational energies and corresponding assigned ν(CN) and ν(CO) modes for the Hox–COexo clusters.

Mentions: The vibrational energies and corresponding ν(CN) and ν(CO) mode assignments are provided for each Hox–COexo cluster from active CpI produced with either natural abundance or isotopically labeled tyrosine. Energies were determined from spectra measured using FTIR spectroscopy (Fig. 4).


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)

Infrared spectra for CpI hydrogenase with isotopically labeled H-cluster containing exogenously bound CO.The infrared spectra are for the CO-inhibited CpI enzyme harboring an 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). Natural abundance COexo was added to CpItyr and CpI2-13C-tyr, which have intrinsic CO ligands. Conversely, 13COexo was added to CpI1-13C-tyr and CpIU-13C-15N-tyr, which have intrinsic 13CO ligands. Comparing the Hox–COexo spectrum for each CpI sample to its respective Hox spectrum (Fig. 3), shifts of 5–10 cm−1 were observed for the ν(CN) modes and the ν(μ–CO) mode in all four cases. The ν(CO) mode for the Fep–CO ligand did not change. Meanwhile, the ν(CO) mode for the Fed–CO moiety was replaced with two peaks resulting from symmetric and asymmetric coupled vibrational stretches, as two CO molecules of equal mass are coordinated to the Fed atom. The peak for the ν(CO)symmetric mode is visible at 2015/1970 cm−1 for CO/13CO. The ν(CO)asymmetric mode, however, cannot be distinguished because its vibrational energy is similar to the ν(CO) mode at 1972/1928 cm−1 for the Fep–CO/Fep–13CO adducts. The changes in vibrational energies, indicated by the dashed lines, correlate with expected changes for ν(13CO), ν(13CN), and ν(13C15N) modes, again confirming that the CO and CN− ligands are synthesized from tyrosine. Labels indicating the assigned ν(CO) and ν(CN) vibrational modes are provided. The 13CN/13C15N and 13CO ligands are shown in red and green, respectively, in the molecular diagrams. Vertical scale bars shown at 1740 cm−1 represent a difference of 0.5 milliabsorbance units. Table 3 summarizes the vibrational energies and corresponding assigned ν(CN) and ν(CO) modes for the Hox–COexo clusters.
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Related In: Results  -  Collection

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

pone-0020346-g004: Infrared spectra for CpI hydrogenase with isotopically labeled H-cluster containing exogenously bound CO.The infrared spectra are for the CO-inhibited CpI enzyme harboring an 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). Natural abundance COexo was added to CpItyr and CpI2-13C-tyr, which have intrinsic CO ligands. Conversely, 13COexo was added to CpI1-13C-tyr and CpIU-13C-15N-tyr, which have intrinsic 13CO ligands. Comparing the Hox–COexo spectrum for each CpI sample to its respective Hox spectrum (Fig. 3), shifts of 5–10 cm−1 were observed for the ν(CN) modes and the ν(μ–CO) mode in all four cases. The ν(CO) mode for the Fep–CO ligand did not change. Meanwhile, the ν(CO) mode for the Fed–CO moiety was replaced with two peaks resulting from symmetric and asymmetric coupled vibrational stretches, as two CO molecules of equal mass are coordinated to the Fed atom. The peak for the ν(CO)symmetric mode is visible at 2015/1970 cm−1 for CO/13CO. The ν(CO)asymmetric mode, however, cannot be distinguished because its vibrational energy is similar to the ν(CO) mode at 1972/1928 cm−1 for the Fep–CO/Fep–13CO adducts. The changes in vibrational energies, indicated by the dashed lines, correlate with expected changes for ν(13CO), ν(13CN), and ν(13C15N) modes, again confirming that the CO and CN− ligands are synthesized from tyrosine. Labels indicating the assigned ν(CO) and ν(CN) vibrational modes are provided. The 13CN/13C15N and 13CO ligands are shown in red and green, respectively, in the molecular diagrams. Vertical scale bars shown at 1740 cm−1 represent a difference of 0.5 milliabsorbance units. Table 3 summarizes the vibrational energies and corresponding assigned ν(CN) and ν(CO) modes for the Hox–COexo clusters.
Mentions: The vibrational energies and corresponding ν(CN) and ν(CO) mode assignments are provided for each Hox–COexo cluster from active CpI produced with either natural abundance or isotopically labeled tyrosine. Energies were determined from spectra measured using FTIR spectroscopy (Fig. 4).

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