(228o) Solution Phase Characterization of [Fe-Fe] Hydrogenase O2 Sensitivity

[FeFe] hydrogenases can provide large specific activities for sustainable, biological production of H₂ but are highly O₂ sensitive. 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 (Fds). 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 assay. The second assay validates the more convenient NADPH-driven assay which also 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 Fds to O₂. However, when decay in H₂ production rate is only assessed after O₂ addition, both assays indicate first order hydrogenase inactivation with rate constants insensitive to initial hydrogenase concentration. A protein fusion between Cp ferredoxin (CpFd) and CpI mediated by a 15 amino acid linker provides a higher specific activity than CpI alone (suggesting more effective electron delivery) and also was more O₂ tolerant. A longer linker, however, provided no activity or O₂ tolerance benefit. 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.