Elucidating in vitro activation of [FeFe] hydrogenase
Abstract/Contents
- Abstract
- The sustainable production of hydrogen for use as a renewable energy source is a promising alternative to current fossil fuel-based technologies. We seek to exploit nature's prolific biological catalysts, hydrogenases, to replace fossil fuel-based hydrogen production with a carbon-neutral process. [FeFe] hydrogenases are specific and productive enzymes for hydrogen evolution, but the application of these complex proteins is limited by inefficient production methods. Here, we present progress towards a fully defined in vitro hydrogenase maturation system by elucidating the assembly requirements for producing fully active [FeFe] hydrogenase. The in vitro [FeFe] hydrogenase maturation system activates purified Clostridium pasteurianum CpI apoprotein using recombinant Shewanella oneidensis HydE, HydF, and HydG maturases, and exogenous small molecules. However, activation can only be achieved in the presence of a crude and undefined E. coli cell extract. In order to identify the protein(s) in the crude cell extract responsible for maturing the hydrogenase, various fractionation and protein separation techniques were implemented, and candidate proteins were evaluated for apo hydrogenase activation. For instance, prokaryotic frataxin, CyaY, interacts with iron sulfur cluster (isc) proteins and, when complexed with ferric iron fully enables activation of the hydrogenase while reducing the required amount of crude extract from 20% to 2% (v/v). Protease treatments were pursued to further separate protein fractions that retained hydrogenase-activating functionality. A 1hr, 60°C papain treatment was effective in significantly reducing proteomic content while maintaining the protein(s) responsible for activating the hydrogenase. After combining papain treatment with chromatography and LC-MS/MS analysis, the identified proteins suggested that oxidoreductases to play an essential role in donating electrons to the maturases. We present a thioredoxin based electron delivery system to active the first fully defined hydrogenase maturation system, requiring super-physiological thioredoxin concentrations. Finally, Ferrous transporter protein A, FeoA, was determined to be a promising candidate for fully maturing the hydrogenase by providing an electron delivery mechanism for H-cluster assembly and installation at physiological FeoA concentrations. In addition to its putative role as a ferrous iron trafficking protein, we propose a new functionality as an electron donor to satisfy the reduction requirements for the [FeFe] hydrogenase active site. These findings provide a deeper understanding of the mechanism for [FeFe] hydrogenase maturation. Further, this knowledge now enables genetic engineering of E. coli and photosynthetic organisms for the biological production of hydrogen as a clean and renewable alternative fuel.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2016 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | De La Paz, Liliana |
|---|---|
| Associated with | Stanford University, Department of Chemical Engineering. |
| Primary advisor | Swartz, James R |
| Thesis advisor | Swartz, James R |
| Thesis advisor | Dunn, Alexander Robert |
| Thesis advisor | Khosla, Chaitan, 1964- |
| Advisor | Dunn, Alexander Robert |
| Advisor | Khosla, Chaitan, 1964- |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Liliana De La Paz. |
|---|---|
| Note | Submitted to the Department of Chemical Engineering. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2016. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2016 by Liliana De La Paz
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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