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Activity and Oxygen Sensitivity of [Fefe ACTIVITY AND OXYGEN SENSITIVITY OF [FEFE] HYDROGENASES A DISSERTATION SUBMITTED TO THE DEPARTMENT OF CHEMICAL ENGINEERING AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Alyssa Sea Bingham Powell December 2012 © 2012 by Alyssa Sea Bingham Powell. All Rights Reserved. Re-distributed by Stanford University under license with the author. This work is licensed under a Creative Commons Attribution- Noncommercial 3.0 United States License. http://creativecommons.org/licenses/by-nc/3.0/us/ This dissertation is online at: http://purl.stanford.edu/zq272fv5447 ii I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. James Swartz, Primary Adviser I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Alexander Dunn I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Alfred Spormann Approved for the Stanford University Committee on Graduate Studies. Patricia J. Gumport, Vice Provost Graduate Education This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file in University Archives. iii iv Abstract Hydrogenases catalyze the reversible conversion of protons and electrons into + - hydrogen, 2H + 2e ↔ H2. Currently, H2 is mainly used as a chemical feedstock for ammonia synthesis in fertilizer and for petroleum refining. As fossil fuel reserves decrease and our demands for electricity and transportation fuels continue to increase, hydrogen as an alternative fuel source may also become important. Currently, hydrogen is produced from steam reformation of natural gas, a process requiring high temperatures and pressures and generating CO2 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. Most [NiFe] hydrogenases instead have a catalytic bias towards hydrogen oxidation and are also much slower catalysts, limiting their use in hydrogen production technologies. The Swartz lab is especially 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 photosystem I to reduce ferredoxin. A heterologously expressed [FeFe] hydrogenase could then produce H2 using electrons from this reduced ferredoxin. One major challenge for developing this and other biohydrogen production systems is that all [FeFe] hydrogenases are irreversibly inactivated by oxygen. The [FeFe] hydrogenase active site, or consists of a [2Fe] subcluster connected to a [4Fe4S] v subcluster by a cysteinyl sulfur. The [2Fe] subcluster is also ligated by carbon monoxide, cyanide, and dithiol bridging ligands. Oxygen exposure results in inactivation and degradation of this complex active site. In a photosynthetic organism, the O2 produced as a byproduct of photosynthesis will therefore inactive the hydrogenase. The goal of the research described here was to evolve an [FeFe] hydrogenase for improved oxygen tolerance. In this thesis, we describe our progress towards evolution of 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 an improved 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. This mutant had three mutations that allowed it to retain more hydrogen oxidation activity after oxygen exposure than wild-type CpI. Saturation mutagenesis at these three influential sites resulted in further improved mutants. Two of these three influential amino acid sites were located near the accessory Fe-S cluster proximal to the H-cluster, suggesting this cluster may be influential in oxygen sensitivity. The accessory Fe-S clusters of CpI (three [4Fe-4S] clusters and one [2Fe-2S] cluster) are believed to relay electrons between the active site and redox substrates that dock at the protein surface. 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. vi 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. Due to the electron source present in the hydrogen production assays, only the irreversible state was observed with hydrogen production activity. However, for the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii (Cr HydA1), only irreversible inactivation was observed in both the hydrogen oxidation and hydrogen production directions. In addition to enzymatic activity assays, we also studied oxygen inactivation using stopped-flow FTIR spectroscopy. Iron-coordinated carbon monoxide and cyanide ligands have unique vibrational stretches that allow us to study the state of the H-cluster using FTIR spectroscopy. The stopped-flow capabilities allowed us to instantaneously mix buffer containing different concentrations of O2 with CpI or Cr 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, after identifying mutations in CpI suggesting the proximal accessory Fe-S cluster to be influential for oxygen sensitivity, we investigated the role of these accessory Fe-S clusters 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] vii 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. We suggest adapting an in vitro thylakoid-membrane based assay developed by Yacoby et al. (PNAS, 2011) to screen for improved hydrogen production from PSI-reduced ferredoxin in the presence of oxygen. viii Acknowledgments I would like to start off by thanking my advisor, Jim Swartz, for all of his help and support throughout this project. I am grateful that I had the opportunity to join Jim’s lab and work on the biohydrogen project. I appreciate that he always made time to listen to the challenges and progress of my research and give me guidance. I have enjoyed my time working in the Swartz lab and I know I learned a lot that I will take with me throughout my career. I especially thank Jim for his support and advice over these last six months during the thesis-writing process. I would like to acknowledge the other members of my reading committee, Alfred Spormann and Alex Dunn, for their time, support, and advice. I thank Craig Criddle and Steve Cramer for serving on my thesis committee. I would also like to acknowledge funding from the Global Climate and Energy Project and the Department of Energy that supported this work. My colleagues have always made the Swartz lab a very enjoyable place to be, and I would like to thank them all for that. Jim Stapleton, Jon Kuchenreuther, and Phil Smith guided me through hydrogenase activity assays, in vivo production and purification, and cell-free protein synthesis. I appreciate the time they spent teaching me when I joined the lab and the opportunities to work with them throughout the years. I would especially like to thank Phil Smith, Stacey Shiigi, and Kunal Mehta for their collaborations and for all the conversations
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