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Title: Characterization of [FeFe] Hydrogenase O2 Sensitivity Using a New, Physiological Approach

Abstract

[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,more » 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.« less

Authors:
; ; ; ;
Publication Date:
Research Org.:
Stanford Univ., CA (United States)
Sponsoring Org.:
USDOE; USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22). Materials Sciences & Engineering Division; National Science Foundation (NSF)
OSTI Identifier:
1769189
Alternate Identifier(s):
OSTI ID: 1535356
Grant/Contract Number:  
FG02–09ER46632; SC0002010
Resource Type:
Published Article
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Name: Journal of Biological Chemistry Journal Volume: 291 Journal Issue: 41; Journal ID: ISSN 0021-9258
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 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.
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Koo, Jamin, Shiigi, Stacey, Rohovie, Marcus, Mehta, Kunal, & Swartz, James R. Characterization of [FeFe] Hydrogenase O2 Sensitivity Using a New, Physiological Approach. United States. https://doi.org/10.1074/jbc.M116.737122
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Koo, Jamin, Shiigi, Stacey, Rohovie, Marcus, Mehta, Kunal, and Swartz, James R. Sat . "Characterization of [FeFe] Hydrogenase O2 Sensitivity Using a New, Physiological Approach". United States. https://doi.org/10.1074/jbc.M116.737122.
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@article{osti_1769189,
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 = {Sat Oct 01 00:00:00 EDT 2016},
month = {Sat Oct 01 00:00:00 EDT 2016}
}
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https://doi.org/10.1074/jbc.M116.737122
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Works referenced in this record:

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

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

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O2-independent formation of the inactive states of NiFe hydrogenase
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[FeFe]-Hydrogenase-Catalyzed H 2 Production in a Photoelectrochemical Biofuel Cell
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Photosynthetic electron partitioning between [FeFe]-hydrogenase and ferredoxin:NADP+-oxidoreductase (FNR) enzymes in vitro
journal, May 2011

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Visible Light-Driven H 2 Production by Hydrogenases Attached to Dye-Sensitized TiO 2 Nanoparticles
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Insulation of a synthetic hydrogen metabolism circuit in bacteria
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Importance of the Protein Framework for Catalytic Activity of [FeFe]-Hydrogenases
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    Works referencing / citing this record:

    O 2 sensitivity and H 2 production activity of hydrogenases—A review
    journal, August 2019

    • Lu, Yuan; Koo, Jamin
    • Biotechnology and Bioengineering, Vol. 116, Issue 11
    • DOI: 10.1002/bit.27136

    Clostridial whole cell and enzyme systems for hydrogen production: current state and perspectives
    journal, November 2018

    • Latifi, Amel; Avilan, Luisana; Brugna, Myriam
    • Applied Microbiology and Biotechnology, Vol. 103, Issue 2
    • DOI: 10.1007/s00253-018-9514-3
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