The Tunnel of Acetyl-Coenzyme A Synthase/Carbon Monoxide Dehydrogenase Regulates Delivery of CO to the Active Site

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Abstract
The effect of [CO] on acetyl-CoA synthesis activity of the isolated α subunit of acetyl-coenzyme A synthase/carbon monoxide dehydrogenase from Moorella thermoacetica was determined. In contrast to the complete α2β2 enzyme where multiple CO molecules exhibit strong cooperative inhibition, α was weakly inhibited, apparently by a single CO with KI = 1.5 ± 0.5 mM; other parameters include kcat = 11 ± 1 min-1 and KM = 30 ± 10 μM. The α subunit lacked the previously described “majority” activity of the complete enzyme but possessed its “residual” activity. The site affording cooperative inhibition may be absent or inoperative in isolated α subunits. Ni-activated α rapidly and reversibly accepted a methyl group from CH3−Co3+FeSP affording the equilibrium constant KMT = 10 ± 4, demonstrating the superior nucleophilicity of αred relative to Co1+FeSP. CO inhibited this reaction weakly (KI = 540 ± 190 μM). NiFeC EPR intensity of α developed in accordance with an apparent Kd = 30 μM, suggesting that the state exhibiting this signal is not responsible for inhibiting catalysis or methyl group transfer and that it may be a catalytic intermediate. At higher [CO], signal intensity declined slightly. Attenuation of catalysis, methyl group transfer, and the NiFeC signal might reflect the same weak CO binding process. Three mutant α2β2 proteins designed to block the tunnel between the A- and C-clusters exhibited little/no activity with CO2 as a substrate and no evidence of cooperative CO inhibition. This suggests that the tunnel was blocked by these mutations and that cooperative CO inhibition is related to tunnel operation. Numerous CO molecules might bind cooperatively to some region associated with the tunnel and institute a conformational change that abolishes the majority activity. Alternatively, crowding of CO in the tunnel may control flow through the tunnel and deliver CO to the A-cluster at the appropriate step of catalysis. Residual activity may involve CO from the solvent binding directly to the A-cluster.