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 describe 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 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(2) 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(2) 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(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.