skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Characterization of [FeFe] Hydrogenase O 2 Sensitivity Using a New, Physiological Approach

Abstract

[FeFe] hydrogenases catalyze rapid H 2 production but are highly O 2 sensitive. Developing O 2-tolerant enzymes is needed for sustainable H 2 production technologies, but the lack of a quantitative and predictive assay for O 2 tolerance has impeded progress. We determine a new approach to provide quantitative assessment of O 2 sensitivity by using an assay employing ferredoxin NADP + reductase (FNR) to transfer electrons from NADPH to hydrogenase via ferredoxins (Fd). Hydrogenase inactivation is measured during H 2 production in an O 2-containing environment. An alternative assay uses dithionite (DTH) to provide reduced Fd. This second assay measures the remaining hydrogenase activity in periodic samples taken from the NADPH-driven reaction sequence. The second assay validates the more convenient NADPH-driven assay which better mimics physiological conditions. During development of the NADPH-driven assay and while characterizing the Clostridium pasteurianum (Cp) [FeFe] hydrogenase, CpI, we detected significant rates of direct electron loss from reduced Fd to O 2. Yet, this loss does not interfere with measurement of first order hydrogenase inactivation, providing rate constants insensitive to initial hydrogenase concentration. We show increased activity and O 2 tolerance for a protein fusion between Cp ferredoxin (CpFd) and CpI mediated by amore » 15 amino acid linker but not for a longer linker. Here, we suggest that this precise, solution phase assay for [FeFe] hydrogenase O 2 sensitivity and the insights we provide constitute an important advance toward the discovery of the O 2 tolerant [FeFe] hydrogenases required for photosynthetic, biological H 2 production.« less

Authors:
 [1];  [1];  [1];  [1];  [1]
  1. Stanford Univ., CA (United States)
Publication Date:
Research Org.:
Stanford Univ., CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22). Materials Sciences & Engineering Division; National Science Foundation (NSF)
OSTI Identifier:
1535356
Grant/Contract Number:  
SC0002010
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 291; Journal Issue: 41; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Country of Publication:
United States
Language:
English
Subject:
enzyme inactivation; enzyme kinetics; fusion protein; hydrogenase; metalloprotein

Citation Formats

Koo, Jamin, Shiigi, Stacey, Rohovie, Marcus, Mehta, Kunal, and Swartz, James R. Characterization of [FeFe] Hydrogenase O2 Sensitivity Using a New, Physiological Approach. United States: N. p., 2016. Web. doi:10.1074/jbc.m116.737122.
Koo, Jamin, Shiigi, Stacey, Rohovie, Marcus, Mehta, Kunal, & Swartz, James R. Characterization of [FeFe] Hydrogenase O2 Sensitivity Using a New, Physiological Approach. United States. doi:10.1074/jbc.m116.737122.
Koo, Jamin, Shiigi, Stacey, Rohovie, Marcus, Mehta, Kunal, and Swartz, James R. Tue . "Characterization of [FeFe] Hydrogenase O2 Sensitivity Using a New, Physiological Approach". United States. doi:10.1074/jbc.m116.737122. https://www.osti.gov/servlets/purl/1535356.
@article{osti_1535356,
title = {Characterization of [FeFe] Hydrogenase O2 Sensitivity Using a New, Physiological Approach},
author = {Koo, Jamin and Shiigi, Stacey and Rohovie, Marcus and Mehta, Kunal and Swartz, James R.},
abstractNote = {[FeFe] hydrogenases catalyze rapid H2 production but are highly O2 sensitive. Developing O2-tolerant enzymes is needed for sustainable H2 production technologies, but the lack of a quantitative and predictive assay for O2 tolerance has impeded progress. We determine a new approach to provide quantitative assessment of O2 sensitivity by using an assay employing ferredoxin NADP+ reductase (FNR) to transfer electrons from NADPH to hydrogenase via ferredoxins (Fd). Hydrogenase inactivation is measured during H2 production in an O2-containing environment. An alternative assay uses dithionite (DTH) to provide reduced Fd. This second assay measures the remaining hydrogenase activity in periodic samples taken from the NADPH-driven reaction sequence. The second assay validates the more convenient NADPH-driven assay which better mimics physiological conditions. During development of the NADPH-driven assay and while characterizing the Clostridium pasteurianum (Cp) [FeFe] hydrogenase, CpI, we detected significant rates of direct electron loss from reduced Fd to O2. Yet, this loss does not interfere with measurement of first order hydrogenase inactivation, providing rate constants insensitive to initial hydrogenase concentration. We show increased activity and O2 tolerance for a protein fusion between Cp ferredoxin (CpFd) and CpI mediated by a 15 amino acid linker but not for a longer linker. Here, we suggest that this precise, solution phase assay for [FeFe] hydrogenase O2 sensitivity and the insights we provide constitute an important advance toward the discovery of the O2 tolerant [FeFe] hydrogenases required for photosynthetic, biological H2 production.},
doi = {10.1074/jbc.m116.737122},
journal = {Journal of Biological Chemistry},
number = 41,
volume = 291,
place = {United States},
year = {2016},
month = {7}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 5 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Development of a synthetic pathway to convert glucose to hydrogen using cell free extracts
journal, August 2015


Orientation-Controlled Electrocatalytic Efficiency of an Adsorbed Oxygen-Tolerant Hydrogenase
journal, November 2015


Generation of hydrogen from NADPH using an [FeFe] hydrogenase
journal, February 2012

  • Smith, Phillip R.; Bingham, Alyssa S.; Swartz, James R.
  • International Journal of Hydrogen Energy, Vol. 37, Issue 3
  • DOI: 10.1016/j.ijhydene.2011.03.172

[FeFe]-Hydrogenase Oxygen Inactivation Is Initiated at the H Cluster 2Fe Subcluster
journal, January 2015

  • Swanson, Kevin D.; Ratzloff, Michael W.; Mulder, David W.
  • Journal of the American Chemical Society, Vol. 137, Issue 5
  • DOI: 10.1021/ja510169s

O2-independent formation of the inactive states of NiFe hydrogenase
journal, November 2012

  • Abou Hamdan, Abbas; Burlat, Bénédicte; Gutiérrez-Sanz, Oscar
  • Nature Chemical Biology, Vol. 9, Issue 1
  • DOI: 10.1038/nchembio.1110

Photosynthetic electron partitioning between [FeFe]-hydrogenase and ferredoxin:NADP+-oxidoreductase (FNR) enzymes in vitro
journal, May 2011

  • Yacoby, I.; Pochekailov, S.; Toporik, H.
  • Proceedings of the National Academy of Sciences, Vol. 108, Issue 23
  • DOI: 10.1073/pnas.1103659108

Visible Light-Driven H 2 Production by Hydrogenases Attached to Dye-Sensitized TiO 2 Nanoparticles
journal, December 2009

  • Reisner, Erwin; Powell, Daniel J.; Cavazza, Christine
  • Journal of the American Chemical Society, Vol. 131, Issue 51
  • DOI: 10.1021/ja907923r

Insulation of a synthetic hydrogen metabolism circuit in bacteria
journal, January 2010

  • Agapakis, Christina M.; Ducat, Daniel C.; Boyle, Patrick M.
  • Journal of Biological Engineering, Vol. 4, Issue 1
  • DOI: 10.1186/1754-1611-4-3

[FeFe]-Hydrogenase-Catalyzed H 2 Production in a Photoelectrochemical Biofuel Cell
journal, February 2008

  • Hambourger, Michael; Gervaldo, Miguel; Svedruzic, Drazenka
  • Journal of the American Chemical Society, Vol. 130, Issue 6
  • DOI: 10.1021/ja077691k

Importance of the Protein Framework for Catalytic Activity of [FeFe]-Hydrogenases
journal, November 2011

  • Knörzer, Philipp; Silakov, Alexey; Foster, Carina E.
  • Journal of Biological Chemistry, Vol. 287, Issue 2
  • DOI: 10.1074/jbc.M111.305797

Hydrogenases for biological hydrogen production
journal, September 2011


Cell-free synthesis and maturation of [FeFe] hydrogenases
journal, January 2008

  • Boyer, Marcus E.; Stapleton, James A.; Kuchenreuther, Jon M.
  • Biotechnology and Bioengineering, Vol. 99, Issue 1
  • DOI: 10.1002/bit.21511

Electrochemical Definitions of O 2 Sensitivity and Oxidative Inactivation in Hydrogenases
journal, December 2005

  • Vincent, Kylie A.; Parkin, Alison; Lenz, Oliver
  • Journal of the American Chemical Society, Vol. 127, Issue 51
  • DOI: 10.1021/ja055160v

High-Yield Expression of Heterologous [FeFe] Hydrogenases in Escherichia coli
journal, November 2010


The oxidative inactivation of FeFe hydrogenase reveals the flexibility of the H-cluster
journal, March 2014

  • Fourmond, Vincent; Greco, Claudio; Sybirna, Kateryna
  • Nature Chemistry, Vol. 6, Issue 4
  • DOI: 10.1038/nchem.1892

Evolution of an [FeFe] hydrogenase with decreased oxygen sensitivity
journal, February 2012

  • Bingham, Alyssa S.; Smith, Phillip R.; Swartz, James R.
  • International Journal of Hydrogen Energy, Vol. 37, Issue 3
  • DOI: 10.1016/j.ijhydene.2011.02.048

Oxygen-Tolerant [NiFe]-Hydrogenases: The Individual and Collective Importance of Supernumerary Cysteines at the Proximal Fe-S Cluster
journal, October 2011

  • Lukey, Michael J.; Roessler, Maxie M.; Parkin, Alison
  • Journal of the American Chemical Society, Vol. 133, Issue 42
  • DOI: 10.1021/ja205393w

Electrochemical Kinetic Investigations of the Reactions of [FeFe]-Hydrogenases with Carbon Monoxide and Oxygen: Comparing the Importance of Gas Tunnels and Active-Site Electronic/Redox Effects
journal, October 2009

  • Goldet, Gabrielle; Brandmayr, Caterina; Stripp, Sven T.
  • Journal of the American Chemical Society, Vol. 131, Issue 41
  • DOI: 10.1021/ja905388j

Protein film voltammetry: electrochemical enzymatic spectroscopy. A review on recent progress
journal, May 2011

  • Gulaboski, Rubin; Mirčeski, Valentin; Bogeski, Ivan
  • Journal of Solid State Electrochemistry, Vol. 16, Issue 7
  • DOI: 10.1007/s10008-011-1397-5

How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms
journal, September 2009

  • Stripp, S. T.; Goldet, G.; Brandmayr, C.
  • Proceedings of the National Academy of Sciences, Vol. 106, Issue 41
  • DOI: 10.1073/pnas.0905343106

How oxygen reacts with oxygen-tolerant respiratory [NiFe]-hydrogenases
journal, April 2014

  • Wulff, P.; Day, C. C.; Sargent, F.
  • Proceedings of the National Academy of Sciences, Vol. 111, Issue 18
  • DOI: 10.1073/pnas.1322393111

Strategies for improving biological hydrogen production
journal, April 2012


Complete activity profile of Clostridium acetobutylicum [FeFe]-hydrogenase and kinetic parameters for endogenous redox partners
journal, October 2007