Chapter Twelve - Production and properties of enzymes that activate and produce carbon monoxide
Introduction
The chapter is focused on the methods involved in producing and characterizing the catalytic properties of the two key nickel iron–sulfur metalloenzymes in the Wood–Ljungdahl pathway (WLP) of anaerobic conversion of CO2 to acetyl-coenzyme A (acetyl-CoA). This pathway involves the eight-electron reduction of CO2 to form acetyl-CoA. In the WLP, these enzymes, carbon monoxide dehydrogenase (CODH), and acetyl-CoA synthase (ACS) intertwine to form a tenacious complex with a 150 Å gas channel that sequesters the CO formed from CO2 (Eq. 1) at the CODH active site and tunnels it to the A-cluster, the active site of ACS, for catalyzing acetyl-CoA synthesis (Eq. 2). The A-cluster binds and then condenses the three components of acetyl-CoA: CO, a methyl group donated by the methyl-Co(III) state of the corrinoid iron–sulfur protein (CFeSP), and coenzyme A. The WLP can operate in reverse to allow organisms to grow on acetate. Furthermore, CODH is a modular unit that can interact with other enzymes, besides ACS, to fulfill various metabolic roles. For example, it forms a CODH/hydrogenase unit to couple CO oxidation to proton reduction. Much progress has been made on understanding the mechanisms of CODH and ACS based on spectroscopic, crystallographic, kinetic, and electrochemical studies.CODH: CO2 + 2H+ + 2 electrons ➔ CO + H2OACS: CO + CH3+ + CoA ➔ CO + CH3-CO-CoA
These enzymes have been purified and characterized from several microbes and are used as important inspirational models and benchmarks in energy technologies ranging from applications such as fuel cells to fundamental examples of “nearly perfect” catalytic activity.
Section snippets
General considerations about technology for growth of anaerobic cultures
Cultivation of anaerobic cell cultures is as ancient as recorded human civilization in the form of fermentation, and it was on facultative anaerobes that the foundational work of Leeuwenhoek and Pasteur was performed, ushering in the age of modern biochemistry. Anaerobic conditions are essential for culturing and harvesting the oxygen-sensitive microbes, including Moorella thermoacetica, Clostridium autoethanogenum, Carboxydothermus hydrogenoformans, and Methanothermobacter marburgensis,
General anaerobic technology
General anaerobic protocols for culturing the microbes were explained earlier, but they are also relevant here. Here we will describe procedures for purifying and handling extremely oxygen-sensitive enzymes. All protein purification steps take place under anoxic conditions in anaerobic chambers (earlier) and reducing agents, usually DTT, Ti(III) citrate, and/or dithionite, are used in all solutions to poise and maintain the low redox potentials required for optimal enzyme activity (Wang, Can,
Characterization of CODH
Anaerobic conditions must be used during all experiments to preserve the active states of the clusters and to poise the solutions at the optimal redox condition for the reaction. All bacterial Ni-CODHs, whether they are monofunctional or bound to ACS, form a homodimeric complex containing five iron–sulfur clusters. Each monomer contains a NiFe3S4 cluster (C-cluster) and a Fe4S4 cluster (B-cluster); in addition, a single Fe4S4 cluster (D-cluster) links the two monomers covalently (see earlier).
Closing and future directions
This is an opportune time to review the methods used to characterize the two key enzymes in the WLP. We now have heterologous and homologous genetic systems for expressing CODH and ACS. Strains of acetogens (Clostridium ljungdahlii, Clostridium acetogenum, Clostridium ragsdalii, etc.) have been developed for biotechnological applications. Combining mutagenesis and directed evolution with mechanistic studies offer a powerful toolbox for gaining a better understanding of the mechanisms of these
Acknowledgments
Work in the Ragsdale lab on the Wood–Ljungdahl Pathway has been funded by NIH (R37-GM39451). S.W.R. is personally grateful to all members of the Ragsdale lab and all our lineages for the progress on understanding the mechanisms of CODH and ACS and the WLP in general.
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