Tight Coupling of Partial Reactions in the Acetyl-CoA Decarbonylase/Synthase (ACDS) Multienzyme Complex from Methanosarcina thermophila

摘要

Direct synthesis and cleavage of acetyl-CoA are carried out by the bifunctional CO dehydrogenase/acetyl-CoA synthase enzyme in anaerobic bacteria and by the acetyl-CoA decarbonylase/synthase (ACDS) multienzyme complex in Archaea. In both systems, a nickel- and Fe/S-containing active site metal center, the A cluster, catalyzes acetyl C–C bond formation/breakdown. Carbonyl group exchange of [1-14C]acetyl-CoA with unlabeled CO, a hallmark of CODH/ACS, is weakly active in ACDS, and exchange with CO2 was up to 350 times faster, indicating tight coupling of CO release at the A cluster to CO oxidation to CO2 at the C cluster in CO dehydrogenase. The basis for tight coupling was investigated by analysis of three recombinant A cluster proteins, ACDS β subunit from Methanosarcina thermophila, acetyl-CoA synthase of Carboxydothermus hydrogenoformans (ACSCh), and truncated ACSCh lacking its 317-amino acid N-terminal domain. A comparison of acetyl-CoA synthesis kinetics, CO exchange, acetyltransferase, and A cluster Ni+-CO EPR characteristics demonstrated a direct role of the ACS N-terminal domain in promoting acetyl C–C bond fragmentation. Protein conformational changes, related to “open/closed” states previously identified crystallographically, were indicated to have direct effects on the coordination geometry and stability of the A cluster Ni2+-acetyl intermediate, controlling Ni2+-acetyl fragmentation and Ni2+(CO)(CH3) condensation. EPR spectral changes likely reflect variations in the Ni+-CO equatorial coordination environment in closed buried hydrophobic and open solvent-exposed states. The involvement of subunit-subunit interactions in ACDS, versus interdomain contacts in ACS, ensures that CO is not released from the ACDS β subunit in the absence of appropriate interactions with the α2?2 CO dehydrogenase component. The resultant high efficiency CO transfer explains the low rate of CO exchange relative to CO2.