Discovery of FeFe hydrogenase variants with enhanced O2 tolerance
Abstract/Contents
- Abstract
- Hydrogenases catalyze the reversible formation of H2 from two protons and two electrons. They are classified as [FeFe], [NiFe], or [Fe]-only with respect to the metal atoms included in the active site. Among the three, [FeFe] hydrogenases have a catalytic bias toward H2 formation. The most prolific enzymes require overpotentials lower than that for platinum, one of the best metal catalysts reported to date. Consequently, [FeFe] hydrogenases offer significant potential for biological production of H2. We have approached such applications with a particular focus on the [FeFe] hydrogenase from Clostridium pasteurianum (CpI). It is a 63.8 kDa protein with one of the highest reported H2 production-specific activities in the order of 1,500 s-1 (or an order of magnitude higher when coupled with an electrode). However, it is also a complex enzyme with three accessory [4Fe-4S] clusters and one [2Fe-2S] cluster that deliver electrons to or from the active site consisting of an [FeFe] sub-cluster bridged to a [4Fe-4S] cluster by a cysteinyl thiol. Much work has been done during the past decade to enable the heterologous expression, maturation, and purification of the active form of CpI in Escherichia coli. Based on these advances, we are now working on two distinct routes of biological H2 production using this enzyme: Fermentative and photosynthetic. Unfortunately, [FeFe] hydrogenases are highly sensitive to O2, imposing a significant technical barrier for biological H2 production. The half-life of CpI in air-saturated buffer is estimated to be only 2--3 min. We worked on three projects to overcome this limitation with the end goal of engineering more O2-tolerant CpI variants. We first developed physiological assays for characterizing the O2 sensitivity of [FeFe] hydrogenases. Next, we developed a highthroughput screening platform for measuring H2 production rates in a biochemical context, including these assays. Empowered by the results of these projects, we evaluated over 300 CpI mutants and discovered several mutations that significantly impact the O2 tolerance of the enzyme. The best combination of mutations enabled greater than 4-fold improvement in aerobic (5.0 vol% O2) H2 yield over an hour. Our work provide the tools that allow convenient assessment of the O2 tolerance of [FeFe] hydrogenases and engineered mutants. The throughput has been improved by more than 10-fold. More importantly, we showed the first successful case of engineering [FeFe] hydrogenases with higher O2 tolerance, further promoting the research towards biological production of H2 for use as a renewable fuel and chemical.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Copyright date | 2017 |
| Publication date | 2016, 2017; 2016 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Koo, Jamin |
|---|---|
| Associated with | Stanford University, Department of Chemical Engineering. |
| Primary advisor | Swartz, James R |
| Thesis advisor | Swartz, James R |
| Thesis advisor | Cochran, Jennifer R |
| Thesis advisor | Dunn, Alexander Robert |
| Advisor | Cochran, Jennifer R |
| Advisor | Dunn, Alexander Robert |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Jamin Koo. |
|---|---|
| Note | Submitted to the Department of Chemical Engineering. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2017. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2017 by Jamin Koo
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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