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Title: The binuclear cluster of [FeFe] hydrogenase is formed with sulfur donated by cysteine of an [Fe(Cys)(CO) 2 (CN)] organometallic precursor

Abstract

The enzyme [FeFe]-hydrogenase (HydA1) contains a unique 6-iron cofactor, the H-cluster, that has unusual ligands to an Fe–Fe binuclear subcluster: CN , CO, and an azadithiolate (adt) ligand that provides 2 S bridges between the 2 Fe atoms. In cells, the H-cluster is assembled by a collection of 3 maturases: HydE and HydF, whose roles aren’t fully understood, and HydG, which has been shown to construct a [Fe(Cys)(CO) 2 (CN)] organometallic precursor to the binuclear cluster. Here, we report the in vitro assembly of the H-cluster in the absence of HydG, which is functionally replaced by adding a synthetic [Fe(Cys)(CO) 2 (CN)] carrier in the maturation reaction. The synthetic carrier and the HydG-generated analog exhibit similar infrared spectra. The carrier allows HydG-free maturation to HydA1, whose activity matches that of the native enzyme. Maturation with 13 CN-containing carrier affords 13 CN-labeled enzyme as verified by electron paramagnetic resonance (EPR)/electron nuclear double-resonance spectra. This synthetic surrogate approach complements existing biochemical strategies and greatly facilitates the understanding of pathways involved in the assembly of the H-cluster. As an immediate demonstration, we clarify that Cys is not the source of the carbon and nitrogen atoms in the adt ligand using pulse EPRmore » to target the magnetic couplings introduced via a 13 C 3 , 15 N-Cys–labeled synthetic carrier. Parallel mass-spectrometry experiments show that the Cys backbone is converted to pyruvate, consistent with a cysteine role in donating S in forming the adt bridge. This mechanistic scenario is confirmed via maturation with a seleno-Cys carrier to form HydA1–Se, where the incorporation of Se was characterized by extended X-ray absorption fine structure spectroscopy.« less

Authors:
ORCiD logo; ; ; ORCiD logo; ORCiD logo; ORCiD logo
Publication Date:
Sponsoring Org.:
USDOE
OSTI Identifier:
1567868
Grant/Contract Number:  
AC02-76SF00515
Resource Type:
Published Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Volume: 116 Journal Issue: 42; Journal ID: ISSN 0027-8424
Publisher:
Proceedings of the National Academy of Sciences
Country of Publication:
United States
Language:
English

Citation Formats

Rao, Guodong, Pattenaude, Scott A., Alwan, Katherine, Blackburn, Ninian J., Britt, R. David, and Rauchfuss, Thomas B. The binuclear cluster of [FeFe] hydrogenase is formed with sulfur donated by cysteine of an [Fe(Cys)(CO) 2 (CN)] organometallic precursor. United States: N. p., 2019. Web. doi:10.1073/pnas.1913324116.
Rao, Guodong, Pattenaude, Scott A., Alwan, Katherine, Blackburn, Ninian J., Britt, R. David, & Rauchfuss, Thomas B. The binuclear cluster of [FeFe] hydrogenase is formed with sulfur donated by cysteine of an [Fe(Cys)(CO) 2 (CN)] organometallic precursor. United States. doi:10.1073/pnas.1913324116.
Rao, Guodong, Pattenaude, Scott A., Alwan, Katherine, Blackburn, Ninian J., Britt, R. David, and Rauchfuss, Thomas B. Mon . "The binuclear cluster of [FeFe] hydrogenase is formed with sulfur donated by cysteine of an [Fe(Cys)(CO) 2 (CN)] organometallic precursor". United States. doi:10.1073/pnas.1913324116.
@article{osti_1567868,
title = {The binuclear cluster of [FeFe] hydrogenase is formed with sulfur donated by cysteine of an [Fe(Cys)(CO) 2 (CN)] organometallic precursor},
author = {Rao, Guodong and Pattenaude, Scott A. and Alwan, Katherine and Blackburn, Ninian J. and Britt, R. David and Rauchfuss, Thomas B.},
abstractNote = {The enzyme [FeFe]-hydrogenase (HydA1) contains a unique 6-iron cofactor, the H-cluster, that has unusual ligands to an Fe–Fe binuclear subcluster: CN − , CO, and an azadithiolate (adt) ligand that provides 2 S bridges between the 2 Fe atoms. In cells, the H-cluster is assembled by a collection of 3 maturases: HydE and HydF, whose roles aren’t fully understood, and HydG, which has been shown to construct a [Fe(Cys)(CO) 2 (CN)] organometallic precursor to the binuclear cluster. Here, we report the in vitro assembly of the H-cluster in the absence of HydG, which is functionally replaced by adding a synthetic [Fe(Cys)(CO) 2 (CN)] carrier in the maturation reaction. The synthetic carrier and the HydG-generated analog exhibit similar infrared spectra. The carrier allows HydG-free maturation to HydA1, whose activity matches that of the native enzyme. Maturation with 13 CN-containing carrier affords 13 CN-labeled enzyme as verified by electron paramagnetic resonance (EPR)/electron nuclear double-resonance spectra. This synthetic surrogate approach complements existing biochemical strategies and greatly facilitates the understanding of pathways involved in the assembly of the H-cluster. As an immediate demonstration, we clarify that Cys is not the source of the carbon and nitrogen atoms in the adt ligand using pulse EPR to target the magnetic couplings introduced via a 13 C 3 , 15 N-Cys–labeled synthetic carrier. Parallel mass-spectrometry experiments show that the Cys backbone is converted to pyruvate, consistent with a cysteine role in donating S in forming the adt bridge. This mechanistic scenario is confirmed via maturation with a seleno-Cys carrier to form HydA1–Se, where the incorporation of Se was characterized by extended X-ray absorption fine structure spectroscopy.},
doi = {10.1073/pnas.1913324116},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 42,
volume = 116,
place = {United States},
year = {2019},
month = {9}
}

Journal Article:
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DOI: 10.1073/pnas.1913324116

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