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

[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.