The central enzyme of the Wood pathway, carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) (previously known only as CODH), catalyses the two reactions:
ACS isolated from M. thermoacetica is a 310 kDa* α2β2 tetramer (it is made of four strands of protein: two identical strands called 'alpha' and two called 'beta'). Each αβ dimer contains three metal-sulfur clusters called A, B, and C. The A-cluster is located in the 81 kDa α subunit while the B- and C-clusters are found in the 71 kDa β subunit.
The A Cluster is where the ACS activity happens:
[CH3-Co3+FeS]n+ + CO + CoASH
-> CH3C(O)SCoA + [Co1+FeS](n-1)+ + H+
This is the reaction that is so similar to the genesis reaction. The methyl group is brought to the enzyme by the Co-containing corrin protein as Co-CH3 (this is the other enzyme with an organometallic bond). The CO comes directly from the C cluster through a tunnel in the enzyme. Click here for a description of the mechanism at the A cluster (which is seen below).
The B cluster is near the C cluster, and provides electrons to the C cluster during the CO2 reduction reaction. The B cluster is a Fe4S4 cube:
The C cluster is where the CO2 is reduced:
CO2 + 2H+ + 2e- -> CO + H2O
The carbon dioxide comes from the environment, and one electron comes from the C cluster itself, and the other comes from the B cluster. The carbon monoxide, harmful to the organism is great concentration, is not allowed to escape back into the cell cytoplasm; instead it is shuttled directly to the A cluster through a molecular tunnel in the enzyme. Click here for a more detailed description of how the C cluster (seen below) works.
The enzyme is purified from the cell cytoplasm using at least four chromatographic steps with a lot of activity assays to keep track of where the enzyme is. Since it is easily damaged by oxygen, all steps are performed in an anaerobic box (less than 1 ppm oxygen!). We can work with our enzyme at room temperature (it's a mesophile, so the protein is naturally protected against mild thermal damage), but must keep all oxidants away. We store our samples in liquid nitrogen. The samples we use are a dark brown solution of 90%+ enzyme.
*kDa stands for kiloDalton, the biochemists way of saying kilogram/mole.
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