470663 Solution Phase Characterization of [Fe-Fe] Hydrogenase O2 Sensitivity

Monday, November 14, 2016
Grand Ballroom B (Hilton San Francisco Union Square)
Jamin Koo1, Stacey A. Shiigi2, Kunal Mehta3, Marcus Rohovie1 and James R. Swartz4, (1)Chemical Engineering, Stanford University, Stanford, CA, (2)Department of Bioengineering, Stanford University, Stanford, CA, (3)Bioengineering, Stanford University, Stanford, CA, (4)Department of Chemical Engineering, Stanford University, Stanford, CA

[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.

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See more of this Session: Poster Session: Bioengineering
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division