Date of Award


Document Type

Open Access Master's Thesis

Degree Name

Master of Science in Biological Sciences (MS)

Administrative Home Department

Department of Biological Sciences

Advisor 1

Stephen Techtmann

Committee Member 1

Rupali Datta

Committee Member 2

Amy Marcarelli


The Nickel based Carbon Monoxide Dehydrogenase (CODH) is an anaerobic metalloenzyme responsible for the reversible conversion of CO and water into CO2 and 2 protons and 2 electrons. This enzyme has importance in the environment as one of Earth’s first carbon fixation pathways, and for human uses as a potential source of biofuels and other commodity chemicals. CODH enzymes are present in a wide array of taxa, many of which are uncultured. In this study we express and purify the catalytic subunit (CooS) of the anaerobic CODH from an uncultured Hydrothermarchaeota JdFR-17 co-expressed with the nickel insertion accessory protein (CooC) from Archaeoglobus fulgidis to generate a CODH complex in E. coli. The protein was then characterized via activity assays and thermal stability assays to test the hypothesis that this CooS was more active for CO oxidation compared to CO2 reduction and functioned at elevated temperatures. A soluble CooS protein was purified that contained both nickel and iron in the active site. This provides evidence that CooC and CooS proteins from two different species can cooperate, and that it is possible to express Archaeal CODH complexes in E. coli. However, enzyme assays yielded inconclusive results. This led us to infer that the CODH complex was expressed, yet inactive.