title: Characterization of [FeFe] Hydrogenase O2 Sensitivity Using a New, Physiological Approach.
abstract: [FeFe] hydrogenases catalyze rapid H2production but are highly O2-sensitive. Developing O2-tolerant enzymes is needed for sustainable H2production technologies, but the lack of a quantitative and predictive assay for O2tolerance has impeded progress. We describe a new approach to provide quantitative assessment of O2sensitivity by using an assay employing ferredoxin NADP+reductase (FNR) to transfer electrons from NADPH to hydrogenase via ferredoxins (Fd). Hydrogenase inactivation is measured during H2production 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 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 O2However, 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 O2tolerance 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 O2sensitivity and the insights we provide constitute an important advance toward the discovery of the O2-tolerant [FeFe] hydrogenases required for photosynthetic, biological H2production.
authors.affiliation: From the Departments of Chemical Engineering and.
authors.forename: Jamin
authors.initials: J
authors.lastname: Koo
authors.affiliation: Bioengineering, Stanford University, Stanford, California 94305.
authors.forename: Stacey
authors.initials: S
authors.lastname: Shiigi
authors.affiliation: From the Departments of Chemical Engineering and.
authors.forename: Marcus
authors.initials: M
authors.lastname: Rohovie
authors.affiliation: Bioengineering, Stanford University, Stanford, California 94305.
authors.forename: Kunal
authors.initials: K
authors.lastname: Mehta
authors.affiliation: From the Departments of Chemical Engineering and jswartz@stanford.edu.|Bioengineering, Stanford University, Stanford, California 94305.
authors.forename: James R
authors.initials: JR
authors.lastname: Swartz
mesh_terms: D003013:Clostridium / Q000201:enzymology*; D005288:Ferredoxins / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry*; D010088:Oxidoreductases / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry*
chemical_list: D005288:Ferredoxins; D006859:Hydrogen; D010088:Oxidoreductases; D010100:Oxygen
keywords: enzyme inactivation; enzyme kinetics; fusion protein; hydrogenase; metalloprotein