Tight coupling of partial reactions in the acetyl-CoA decarbonylase/synthase (ACDS) multienzyme complex from Methanosarcina thermophila: acetyl C-C bond fragmentation at the a cluster promoted by protein conformational changes.
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-(14)C]acetyl-CoA with unlabeled CO, a hallmark of CODH/ACS, is weakly active in ACDS, and exchange with CO(2) was up to 350 times faster, indicating tight coupling of CO release at the A cluster to CO oxidation to CO(2) at the C cluster in CO dehydrogenase. The basis for tight coupling was investigated by analysis of three recombinant A cluster proteins, ACDS beta subunit from Methanosarcina thermophila, acetyl-CoA synthase of Carboxydothermus hydrogenoformans (ACS(Ch)), and truncated ACS(Ch) 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 Ni(2+)-acetyl intermediate, controlling Ni(2+)-acetyl fragmentation and Ni(2+)(CO)(CH(3)) 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 beta subunit in the absence of appropriate interactions with the alpha(2)epsilon(2) CO dehydrogenase component. The resultant high efficiency CO transfer explains the low rate of CO exchange relative to CO(2).
Gencic
Simonida
S
Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814.
Duin
Evert C
EC
Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama 36849.
Grahame
David A
DA
Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814. Electronic address: dgrahame@usuhs.mil.
D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D002248:Carbon Monoxide / Q000378:metabolism; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D017020:Methanosarcina / Q000201:enzymology*; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D002248:Carbon Monoxide; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase