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Title: Electron Spin Relaxation and Biochemical Characterization of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S] Cluster Communication and Hydrogenase Activation

Abstract

Nature utilizes [FeFe]-hydrogenase enzymes to catalyze the interconversion between H 2 and protons and electrons. Catalysis occurs at the H-cluster, a carbon monoxide-, cyanide-, and dithiomethylamine-coordinated 2Fe subcluster bridged via a cysteine to a [4Fe-4S] cluster. Biosynthesis of this unique metallocofactor is accomplished by three maturase enzymes denoted HydE, HydF, and HydG. HydE and HydG belong to the radical S-adenosylmethionine superfamily of enzymes and synthesize the nonprotein ligands of the H-cluster. These enzymes interact with HydF, a GTPase that acts as a scaffold or carrier protein during 2Fe subcluster assembly. Prior characterization of HydF demonstrated the protein exists in both dimeric and tetrameric states and coordinates both [4Fe-4S] 2+/+ and [2Fe-2S] 2+/+ clusters [Shepard, E. M., Byer, A. S., Betz, J. N., Peters, J. W., and Broderick, J. B. (2016) Biochemistry 55, 3514–3527]. Herein, electron paramagnetic resonance (EPR) is utilized to characterize the [2Fe-2S] + and [4Fe-4S]+ clusters bound to HydF. Examination of spin relaxation times using pulsed EPR in HydF samples exhibiting both [4Fe-4S] + and [2Fe-2S] + cluster EPR signals supports a model in which the two cluster types either are bound to widely separated sites on HydF or are not simultaneously bound to a single HydF species.more » Gel filtration chromatographic analyses of HydF spectroscopic samples strongly suggest the [2Fe-2S] + and [4Fe-4S] + clusters are coordinated to the dimeric form of the protein. Lastly, we examined the 2Fe subcluster-loaded form of HydF and showed the dimeric state is responsible for [FeFe]-hydrogenase activation. Together, the results indicate a specific role for the HydF dimer in the H-cluster biosynthesis pathway.« less

Authors:
 [1];  [1];  [2];  [1];  [1];  [1];  [1];  [2];  [2]; ORCiD logo [1]
  1. Montana State Univ., Bozeman, MT (United States). Dept. of Chemistry and Biochemistry
  2. Univ. of Denver, CO (United States). Dept. of Chemistry and Biochemistry
Publication Date:
Research Org.:
Montana State Univ., Bozeman, MT (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1363692
Alternate Identifier(s):
OSTI ID: 1469949
Grant/Contract Number:  
SC0005404; FG02-10ER16194
Resource Type:
Journal Article: Published Article
Journal Name:
Biochemistry
Additional Journal Information:
Journal Volume: 56; Journal Issue: 25; Journal ID: ISSN 0006-2960
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY

Citation Formats

Shepard, Eric M., Byer, Amanda S., Aggarwal, Priyanka, Betz, Jeremiah N., Scott, Anna G., Shisler, Krista A., Usselman, Robert J., Eaton, Gareth R., Eaton, Sandra S., and Broderick, Joan B. Electron Spin Relaxation and Biochemical Characterization of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S] Cluster Communication and Hydrogenase Activation. United States: N. p., 2017. Web. doi:10.1021/acs.biochem.7b00169.
Shepard, Eric M., Byer, Amanda S., Aggarwal, Priyanka, Betz, Jeremiah N., Scott, Anna G., Shisler, Krista A., Usselman, Robert J., Eaton, Gareth R., Eaton, Sandra S., & Broderick, Joan B. Electron Spin Relaxation and Biochemical Characterization of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S] Cluster Communication and Hydrogenase Activation. United States. doi:10.1021/acs.biochem.7b00169.
Shepard, Eric M., Byer, Amanda S., Aggarwal, Priyanka, Betz, Jeremiah N., Scott, Anna G., Shisler, Krista A., Usselman, Robert J., Eaton, Gareth R., Eaton, Sandra S., and Broderick, Joan B. Fri . "Electron Spin Relaxation and Biochemical Characterization of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S] Cluster Communication and Hydrogenase Activation". United States. doi:10.1021/acs.biochem.7b00169.
@article{osti_1363692,
title = {Electron Spin Relaxation and Biochemical Characterization of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S] Cluster Communication and Hydrogenase Activation},
author = {Shepard, Eric M. and Byer, Amanda S. and Aggarwal, Priyanka and Betz, Jeremiah N. and Scott, Anna G. and Shisler, Krista A. and Usselman, Robert J. and Eaton, Gareth R. and Eaton, Sandra S. and Broderick, Joan B.},
abstractNote = {Nature utilizes [FeFe]-hydrogenase enzymes to catalyze the interconversion between H2 and protons and electrons. Catalysis occurs at the H-cluster, a carbon monoxide-, cyanide-, and dithiomethylamine-coordinated 2Fe subcluster bridged via a cysteine to a [4Fe-4S] cluster. Biosynthesis of this unique metallocofactor is accomplished by three maturase enzymes denoted HydE, HydF, and HydG. HydE and HydG belong to the radical S-adenosylmethionine superfamily of enzymes and synthesize the nonprotein ligands of the H-cluster. These enzymes interact with HydF, a GTPase that acts as a scaffold or carrier protein during 2Fe subcluster assembly. Prior characterization of HydF demonstrated the protein exists in both dimeric and tetrameric states and coordinates both [4Fe-4S]2+/+ and [2Fe-2S]2+/+ clusters [Shepard, E. M., Byer, A. S., Betz, J. N., Peters, J. W., and Broderick, J. B. (2016) Biochemistry 55, 3514–3527]. Herein, electron paramagnetic resonance (EPR) is utilized to characterize the [2Fe-2S]+ and [4Fe-4S]+ clusters bound to HydF. Examination of spin relaxation times using pulsed EPR in HydF samples exhibiting both [4Fe-4S]+ and [2Fe-2S]+ cluster EPR signals supports a model in which the two cluster types either are bound to widely separated sites on HydF or are not simultaneously bound to a single HydF species. Gel filtration chromatographic analyses of HydF spectroscopic samples strongly suggest the [2Fe-2S]+ and [4Fe-4S]+ clusters are coordinated to the dimeric form of the protein. Lastly, we examined the 2Fe subcluster-loaded form of HydF and showed the dimeric state is responsible for [FeFe]-hydrogenase activation. Together, the results indicate a specific role for the HydF dimer in the H-cluster biosynthesis pathway.},
doi = {10.1021/acs.biochem.7b00169},
journal = {Biochemistry},
number = 25,
volume = 56,
place = {United States},
year = {Fri May 19 00:00:00 EDT 2017},
month = {Fri May 19 00:00:00 EDT 2017}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1021/acs.biochem.7b00169

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