Expression, purification, and activation of one key enzyme in anaerobic CO2 fixation: Carbon monoxide dehydrogenase II from Carboxydothermus hydrogenoformans.

Abstract

Climate change due to anthropomorphic emissions will increase global temperature by at least 1.5 °C by the year 2030. One strategy to reduce the severity of the effects of climate change is to sequester carbon dioxide via natural biochemical cycles. Carbon monoxide dehydrogenase (CODH) has the remarkable ability to catalyze the reversible reduction of CO₂ to CO without an overpotential and without reducing protons. It also is a key enzyme in the Wood-Ljungdahl pathway (WLP), which is the only known anaerobic carbon fixation pathway and fixes 10 % of carbon on earth every year. Characterization of this pathway is crucial because it may enable tools to mitigate climate change by using CO₂ to produce biofuels, chemical feedstocks, and polymers. In the WLP, CODH associates with Acetyl-Coenzyme A synthase (ACS), which catalyzes the condensation of CO from CODH, a methyl group from a B₁₂-dependent methyltransferase, and CoA to form acetyl-CoA. In this complex, CO is shuttled through a 138 Å gas tunnel between the two enzymes. One valuable model for studying the CODH component of CODH/ACS is CODH-II from Carboxydothermus hydrogenoformans because it is stand-alone and is conducive to recombinant expression. Here we describe a detailed protocol for producing high-activity CODH-II in E. coli.