Activity and oxygen sensitivity of [FeFe] hydrogenases
Abstract/Contents
- Abstract
- Hydrogenases catalyze the reversible conversion of protons and electrons into hydrogen. Currently, hydrogen is mainly used as a chemical feedstock for ammonia synthesis for fertilizer and for petroleum refining, but hydrogen as an alternative fuel source may also become important. Hydrogen is produced primarily from steam reformation of natural gas, a process requiring high temperatures and pressures and generating carbon dioxide as a byproduct. Instead, we need a renewable and carbon-neutral process, and hydrogenases are promising catalysts for use in such technologies. [FeFe] hydrogenases are especially of interest for biohydrogen production technologies as they have amazingly fast catalytic rates and tend to work in the hydrogen production direction. The Swartz lab is interested in expressing an [FeFe] hydrogenase in an engineered cyanobacterium to produce hydrogen from sunlight and water. In this organism, water splitting at photosystem II would generate electrons, protons, and oxygen. These electrons would be directed through ferredoxin to a heterologously expressed [FeFe] hydrogenase for hydrogen production. One major challenge for developing this and other biohydrogen production systems is that oxygen exposure results in inactivation and degradation of the complex [FeFe] hydrogenase active site. In a photosynthetic organism, the oxygen produced as a byproduct of photosynthesis will therefore inactive the hydrogenase. In this thesis, we describe our progress towards evolution of an [FeFe] hydrogenase for improved oxygen tolerance and insights we gained into [FeFe] hydrogenase activity and oxygen inactivation. We first evaluated a mutant hydrogenase library for improved oxygen tolerance using a cell-free protein synthesis plate-based screening platform previously developed by Stapleton and Swartz (PLoS ONE, 2010). From a randomly mutated library of Clostridium pasteurianum hydrogenase I (CpI), we identified a mutant with decreased oxygen sensitivity. Saturation mutagenesis at three influential sites resulted in further improved mutants. However, we found that while these mutants were significantly less sensitive to oxygen in the hydrogen oxidation assay (which measured hydrogenase-catalyzed methyl viologen reduction rates), they were actually slightly more sensitive to oxygen than wild-type CpI when hydrogen production was measured. Further studies suggested two inactive states of CpI: one reversible and one irreversible. The reversible inactivated state reactivates slowly in the presence of hydrogen and the electron acceptor methyl viologen, or rapidly in the presence of reducing agents such as dithionite and reduced ferredoxin. The irreversible inactive state does not recover activity. In addition to enzymatic activity assays, we also studied oxygen inactivation using stopped-flow Fourier transform infrared (FTIR) spectroscopy. The stopped-flow capabilities allowed us to mix buffer containing different concentrations of oxygen with CpI or Chlamydomonas reinhardtii HydA1 and obtain kinetic FTIR spectra over the course of the reaction. We discuss possible mechanisms of oxygen inactivation based on both the enzymatic activity assays and spectroscopic investigations. Finally, we investigated the role of the accessory Fe-S clusters of CpI in electron transfer between the H-cluster and various redox substrates. By mutagenesis of the ligating cysteine residues, we generated CpI mutants lacking assembly of one or more accessory Fe-S clusters. We found that electron transfer between CpI and both the Synechocystis [2Fe-2S] ferredoxin and the Clostridium pasteurianum 2[4Fe-4S] ferredoxin occurs mainly through the distal [4Fe-4S] accessory cluster. However, electron transfer between CpI and methyl viologen occurs to a significant extent at both the distal [4Fe-4S] and distal [2Fe-2S] accessory clusters, and to a smaller extent with the surface-inaccessible Fe-S clusters or H-cluster. Overall in this thesis, we describe significant insights gained into both the activity and oxygen sensitivity of [FeFe] hydrogenases. We identified a mutant less sensitive to oxygen in the hydrogen oxidation but not hydrogen production direction. We observed that both the activity and oxygen tolerance are dependent on the assay used to measure them. We conclude that in order to evolve an oxygen-tolerant hydrogenase for photosynthetic hydrogen production, the enzyme should be screened for oxygen tolerance in the hydrogen production direction and with the desired redox partner, ferredoxin.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2012 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Powell, Alyssa Sea Bingham |
|---|---|
| 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 | Spormann, Alfred M |
| Advisor | Dunn, Alexander Robert |
| Advisor | Spormann, Alfred M |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Alyssa Sea Bingham Powell. |
|---|---|
| Note | Submitted to the Department of Chemical Engineering. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2012. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2012 by Alyssa Sea Bingham Powell
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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