Publications

• Nickel in subunit beta of the acetyl-CoA decarbonylase/synthase multienzyme complex in methanogens. Catalytic properties and evidence for a binuclear Ni-Ni site.

  Gencic S., Grahame D.A.

  The acetyl-CoA decarbonylase/synthase (ACDS) complex catalyzes the central reaction of acetyl C-C bond cleavage in methanogens growing on acetate and is also responsible for synthesis of acetyl units during growth on C-1 substrates. The ACDS beta subunit contains nickel and an Fe/S center and reac ... >> More

  The acetyl-CoA decarbonylase/synthase (ACDS) complex catalyzes the central reaction of acetyl C-C bond cleavage in methanogens growing on acetate and is also responsible for synthesis of acetyl units during growth on C-1 substrates. The ACDS beta subunit contains nickel and an Fe/S center and reacts with acetyl-CoA forming an acetyl-enzyme intermediate presumably directly involved in acetyl C-C bond activation. To investigate the role of nickel in this process two forms of the Methanosarcina thermophila beta subunit were overexpressed in anaerobically grown Escherichia coli. Both contained an Fe/S center but lacked nickel and were inactive in acetyl-enzyme formation in redox-dependent acetyltransferase assays. However, high activity developed during incubation with NiCl(2). The native and nickel-reconstituted proteins both contained iron and nickel in a 2:1 ratio, with insignificant levels of other metals, including copper. Binding of nickel elicited marked changes in the UV-visible spectrum, with intense charge transfer bands indicating multiple thiolate ligation to nickel. The kinetics of nickel incorporation matched the time course for enzyme activation. Other divalent metal ions could not substitute for nickel in yielding catalytic activity. Acetyl-CoA was formed in reactions with CoA, CO, and methylcobalamin, directly demonstrating C-C bond activation by the beta subunit in the absence of other ACDS subunits. Nickel was indispensable in this process too and was needed to form a characteristic EPR-detectable enzyme-carbonyl adduct in reactions with CO. In contrast to enzyme activation, EPR signal formation did not require addition of reducing agent, indicating indirect catalytic involvement of the paramagnetic species. Site-directed mutagenesis indicated that Cys-278 and Cys-280 coordinate nickel, with Cys-189 essential for Fe/S cluster formation. The results are consistent with an Ni(2)[Fe(4)S(4)] arrangement at the active site. A mechanism for C-C bond activation is proposed that includes a specific role for the Fe(4)S(4) center and accounts for the absolute requirement for nickel. << Less

  J. Biol. Chem. 278:6101-6110(2003) [PubMed] [EuropePMC]

• Acetate biosynthesis by acetogenic bacteria. Evidence that carbon monoxide dehydrogenase is the condensing enzyme that catalyzes the final steps of the synthesis.

  Ragsdale S.W., Wood H.G.

  The purified carbon monoxide dehydrogenase from Clostridium thermoaceticum is the only protein required to catalyze an exchange reaction between carbon monoxide and the carbonyl group of acetyl-CoA. This exchange requires that the CO dehydrogenase bind the methyl, the carbonyl, and the CoA groups ... >> More

  The purified carbon monoxide dehydrogenase from Clostridium thermoaceticum is the only protein required to catalyze an exchange reaction between carbon monoxide and the carbonyl group of acetyl-CoA. This exchange requires that the CO dehydrogenase bind the methyl, the carbonyl, and the CoA groups of acetyl-CoA, then equilibrate the carbonyl with CO in the solution and re-form acetyl-CoA. CoA is not necessary for the exchange and, in fact, inhibits the reaction. These studies support the view that CO dehydrogenase is the condensing enzyme that forms acetyl-CoA from its component parts. Carbon dioxide also exchanges with the C-1 of acetyl-CoA, but at a much lower rate than does CO. At 50 degrees C and pH 5.3, the optimal pH, the turnover number is 70 mol of CO exchanged per min/mol of enzyme. Low potential electron carriers are stimulatory. The Km app for stimulation by ferredoxin is 50-fold less than the value for flavodoxin. Neither ATP or Pi stimulate the exchange. The EPR spectrum of the CO-reacted enzyme is markedly changed by binding of CoA or acetyl-CoA. Arginine residues of the CO dehydrogenase appear to be involved in the active site, possibly by binding acetyl-CoA. Mersalyl acid, methyl iodide, 5,5-dithiobis-(2-nitrobenzoate), and sodium dithionite inhibit the exchange reaction. A scheme is presented to account for the role of CO dehydrogenase in the exchange reaction and in the synthesis of acetate. << Less

  J Biol Chem 260:3970-3977(1985) [PubMed] [EuropePMC]

• Acetyl-CoA decarbonylase/synthase complex from Archaeoglobus fulgidus.

  Dai Y.R., Reed D.W., Millstein J.H., Hartzell P.L., Grahame D.A., DeMoll E.

  The acetyl-CoA decarbonylase/synthase (ACDS) multienzyme complex catalyzes the reversible cleavage and synthesis of acetyl-CoA in methanogens. This report of the enzyme complex in Archaeoglobus fulgidus demonstrates the existence of a functional ACDS complex in an organism that is not a methanogen ... >> More

  The acetyl-CoA decarbonylase/synthase (ACDS) multienzyme complex catalyzes the reversible cleavage and synthesis of acetyl-CoA in methanogens. This report of the enzyme complex in Archaeoglobus fulgidus demonstrates the existence of a functional ACDS complex in an organism that is not a methanogen. The A. fulgidus enzyme complex contained five subunits of 89, 72, 50, 49.5, and 18.5 kDa, and it catalyzed the overall synthesis of acetyl-CoA according to the following reaction: CO2 + 2 Fdred(Fe2+) + 2 H+ + CH3 - H4SPt + CoA <==> acetyl-CoA + H4SPt + 2 Fdox(Fe3+) + H2O where Fd is ferredoxin, and CH3-H4SPt and H4SPt denote N5-methyl-tetrahydrosarcinapterin and tetrahydrosarcinapterin, respectively. << Less

  Arch. Microbiol. 169:525-529(1998) [PubMed] [EuropePMC]

• A Ni-Fe-Cu center in a bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase.

  Doukov T.I., Iverson T.M., Seravalli J., Ragsdale S.W., Drennan C.L.

  A metallocofactor containing iron, sulfur, copper, and nickel has been discovered in the enzyme carbon monoxide dehydrogenase/acetyl-CoA (coenzyme A) synthase from Moorella thermoacetica (f. Clostridium thermoaceticum). Our structure at 2.2 angstrom resolution reveals that the cofactor responsible ... >> More

  A metallocofactor containing iron, sulfur, copper, and nickel has been discovered in the enzyme carbon monoxide dehydrogenase/acetyl-CoA (coenzyme A) synthase from Moorella thermoacetica (f. Clostridium thermoaceticum). Our structure at 2.2 angstrom resolution reveals that the cofactor responsible for the assembly of acetyl-CoA contains a [Fe4S4] cubane bridged to a copper-nickel binuclear site. The presence of these three metals together in one cluster was unanticipated and suggests a newly discovered role for copper in biology. The different active sites of this bifunctional enzyme complex are connected via a channel, 138 angstroms long, that provides a conduit for carbon monoxide generated at the C-cluster on one subunit to be incorporated into acetyl-CoA at the A-cluster on the other subunit. << Less

  Science 298:567-572(2002) [PubMed] [EuropePMC]