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 describe 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 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 O2 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 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. 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.
Highlights
The abbreviations used areCpI, Clostridium pasteurianum [FeFe] hydrogenase I; Cp ferredoxin (CpFd), Clostridium pasteurianum ferredoxin; ferredoxin NADP؉ reductase (FNR), ferredoxin-NADPHreductase; Fd, ferredoxin; Fd in the reduced state (Fdred), reduced ferredoxin; G6P, glucose 6-phosphate; G6PD, glucose-6-phosphate dehydrogenase; SynFd, Synechocystis sp
We have approached such applications with a particular focus on the [FeFe] hydrogenase from Clostridium pasteurianum
We report the development of a convenient assay for assessing [FeFe] hydrogenase O2 sensitivity
Summary
CpI, Clostridium pasteurianum [FeFe] hydrogenase I; CpFd, Clostridium pasteurianum ferredoxin; FNR, ferredoxin-NADPHreductase; Fd, ferredoxin; Fdred, reduced ferredoxin; G6P, glucose 6-phosphate; G6PD, glucose-6-phosphate dehydrogenase; SynFd, Synechocystis sp. The second assay (Fig. 1b) uses a sequence in which electrons are delivered from DTH to hydrogenase via Fd. The second assay (Fig. 1b) uses a sequence in which electrons are delivered from DTH to hydrogenase via Fd In this assay, the strong reducing power of DTH (EoЈ Ϫ0.66 V versus normal hydrogen electrode at pH 7) consumes any remaining O2, and apparently maintains most of the Fd in the reduced state (Fdred), allowing an assessment of the full residual activity of hydrogenase after O2 exposure. The strong reducing power of DTH (EoЈ Ϫ0.66 V versus normal hydrogen electrode at pH 7) consumes any remaining O2, and apparently maintains most of the Fd in the reduced state (Fdred), allowing an assessment of the full residual activity of hydrogenase after O2 exposure Using these assays, we studied the kinetics of inactivation with varying concentrations of O2 and CpI. We show that a CpI-Fd fusion protein with a higher hydrogenase activity is less sensitive to O2 inactivation
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