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Title: H-Cluster Assembly Intermediates Built on HydF by the Radical SAM Enzymes HydE and HydG

Abstract

[FeFe]-hydrogenase catalyzes the reversible reduction of protons to H2 at a complex metallocofactor site, the H-cluster. Biosynthesis of this active-site H-cluster requires three maturation enzymes: the radical S-adenosylmethionine enzymes HydE and HydG synthesize the nonprotein ligands, while the GTPase HydF provides a scaffold for assembly of the 2Fe subcluster of the H-cluster ([2Fe]H) prior to its transfer to hydrogenase. To delineate the assembly and delivery steps for the 2Fe precursor cluster coordinated to HydF ([2Fe]F), we have heterologously expressed HydF in the presence of HydE alone (HydFE) or HydG alone (HydFG), and characterized the resulting purified HydFE and HydFG using UV-visible, EPR, and FTIR spectroscopies and biochemical assays. The iron-sulfur clusters on HydF are modified by co-expression with HydE or HydG, as evidenced by the changes in the visible, EPR, and FTIR spectral features. Further, biochemical assays show that HydFE is capable of activating HydA..delta..EFG to a limited extent (~1% of WT) even though the normal source of CO and CN- ligands of [2Fe]H (HydG) was absent. Activation assays performed with HydFG, in contrast, exhibit no ability to mature HydA..delta..EFG. It appears that in the case of HydFE, trace diatomics from the cellular environment are incorporated into a [2Fe]F-like precursormore » on HydF in the absence of HydG. We conclude that the product of HydE, presumably the dithiomethylamine ligand of [2Fe]H, is absolutely essential to the activation process, while the diatomic products of HydG can be provided from alternate sources.« less

Authors:
 [1];  [1];  [2];  [1]; ORCiD logo [2];  [1];  [1]
  1. Montana State University
  2. National Renewable Energy Laboratory (NREL), Golden, CO (United States)
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1566047
Report Number(s):
NREL/JA-2700-74929
Grant/Contract Number:  
AC36-08GO28308
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Biological Inorganic Chemistry
Additional Journal Information:
Journal Volume: 24; Journal Issue: 6
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; [FeFe] hydrogenase; H-cluster; maturation; biosynthesis; radical SAM; HydF

Citation Formats

Byer, Amanda S., Shepard, Eric M., Ratzloff, Michael W, Betz, Jeremiah N., King, Paul W, Broderick, William E., and Broderick, Joan B. H-Cluster Assembly Intermediates Built on HydF by the Radical SAM Enzymes HydE and HydG. United States: N. p., 2019. Web. doi:10.1007/s00775-019-01709-7.
Byer, Amanda S., Shepard, Eric M., Ratzloff, Michael W, Betz, Jeremiah N., King, Paul W, Broderick, William E., & Broderick, Joan B. H-Cluster Assembly Intermediates Built on HydF by the Radical SAM Enzymes HydE and HydG. United States. doi:10.1007/s00775-019-01709-7.
Byer, Amanda S., Shepard, Eric M., Ratzloff, Michael W, Betz, Jeremiah N., King, Paul W, Broderick, William E., and Broderick, Joan B. Fri . "H-Cluster Assembly Intermediates Built on HydF by the Radical SAM Enzymes HydE and HydG". United States. doi:10.1007/s00775-019-01709-7.
@article{osti_1566047,
title = {H-Cluster Assembly Intermediates Built on HydF by the Radical SAM Enzymes HydE and HydG},
author = {Byer, Amanda S. and Shepard, Eric M. and Ratzloff, Michael W and Betz, Jeremiah N. and King, Paul W and Broderick, William E. and Broderick, Joan B.},
abstractNote = {[FeFe]-hydrogenase catalyzes the reversible reduction of protons to H2 at a complex metallocofactor site, the H-cluster. Biosynthesis of this active-site H-cluster requires three maturation enzymes: the radical S-adenosylmethionine enzymes HydE and HydG synthesize the nonprotein ligands, while the GTPase HydF provides a scaffold for assembly of the 2Fe subcluster of the H-cluster ([2Fe]H) prior to its transfer to hydrogenase. To delineate the assembly and delivery steps for the 2Fe precursor cluster coordinated to HydF ([2Fe]F), we have heterologously expressed HydF in the presence of HydE alone (HydFE) or HydG alone (HydFG), and characterized the resulting purified HydFE and HydFG using UV-visible, EPR, and FTIR spectroscopies and biochemical assays. The iron-sulfur clusters on HydF are modified by co-expression with HydE or HydG, as evidenced by the changes in the visible, EPR, and FTIR spectral features. Further, biochemical assays show that HydFE is capable of activating HydA..delta..EFG to a limited extent (~1% of WT) even though the normal source of CO and CN- ligands of [2Fe]H (HydG) was absent. Activation assays performed with HydFG, in contrast, exhibit no ability to mature HydA..delta..EFG. It appears that in the case of HydFE, trace diatomics from the cellular environment are incorporated into a [2Fe]F-like precursor on HydF in the absence of HydG. We conclude that the product of HydE, presumably the dithiomethylamine ligand of [2Fe]H, is absolutely essential to the activation process, while the diatomic products of HydG can be provided from alternate sources.},
doi = {10.1007/s00775-019-01709-7},
journal = {Journal of Biological Inorganic Chemistry},
number = 6,
volume = 24,
place = {United States},
year = {2019},
month = {9}
}

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Works referenced in this record:

Electron spin relaxation of iron-sulphur proteins studied by microwave power saturation
journal, December 1978

  • Rupp, H.; Rao, K. K.; Hall, D. O.
  • Biochimica et Biophysica Acta (BBA) - Protein Structure, Vol. 537, Issue 2, p. 255-269
  • DOI: 10.1016/0005-2795(78)90509-3

EasySpin, a comprehensive software package for spectral simulation and analysis in EPR
journal, January 2006