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Characterization of [FeFe] Hydrogenase O2 Sensitivity Using a New, Physiological Approach.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2016 Oct 07; Vol. 291 (41), pp. 21563-21570. Date of Electronic Publication: 2016 Jul 19. - Publication Year :
- 2016
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Abstract
- [FeFe] hydrogenases catalyze rapid H <subscript>2</subscript> production but are highly O <subscript>2</subscript> -sensitive. Developing O <subscript>2</subscript> -tolerant enzymes is needed for sustainable H <subscript>2</subscript> production technologies, but the lack of a quantitative and predictive assay for O <subscript>2</subscript> tolerance has impeded progress. We describe a new approach to provide quantitative assessment of O <subscript>2</subscript> sensitivity by using an assay employing ferredoxin NADP <superscript>+</superscript> reductase (FNR) to transfer electrons from NADPH to hydrogenase via ferredoxins (Fd). Hydrogenase inactivation is measured during H <subscript>2</subscript> production in an O <subscript>2</subscript> -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 solutions. 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 <subscript>2</subscript> However, 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 <subscript>2</subscript> tolerance for a protein fusion between Cp ferredoxin (CpFd) and CpI mediated by a 15-amino acid linker but not for a longer linker. We suggest that this precise, solution phase assay for [FeFe] hydrogenase O <subscript>2</subscript> sensitivity and the insights we provide constitute an important advance toward the discovery of the O <subscript>2</subscript> -tolerant [FeFe] hydrogenases required for photosynthetic, biological H <subscript>2</subscript> production.<br /> (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 291
- Issue :
- 41
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 27435671
- Full Text :
- https://doi.org/10.1074/jbc.M116.737122