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An enzyme complex was isolated from acetate-grown Methanosarcina thermophila that oxidized CO and catalyzed the synthesis or cleavage of acetyl-CoA. The complex consisted of five subunits (alllylle) of 89, 71, 60, 58, and 19 kDa. The complex contained nickel, iron, acid-labile sulfide, and cobalt in a corrinoid cofactor. Two components were resolved by anion-exchange chromatography of the complex in the presence of dodecyltrimethylammonium bromide and Triton X-100: a 200-kDa nickel/iron-sulfur protein with the 89-and 19-kDa (aze,) subunits and a 100-kDa corrinoid/ironsulfur protein with the 60-and 58-kDa subunits (yvi8 thermophila. The nickel/iron-sulfur component also oxidized CO and transferred electrons to the corrinold/iron-sulfur component, reducing the iron-sulfur and Co centers. UVvisible spectroscopy indicated that the reduced corrinoid/ironsulfur component could be methylated with CH3I. The results suggest that the enzyme complex from M. thenrophila contained at least two enzyme components, each with a specific function. The properties of the component enzymes support a mechanism proposed for acetyl-CoA synthesis (or cleavage) by the enzyme complex.The methanogenic archaebacteria utilize two major energyyielding pathways. The more extensively studied pathway is the oxidation of H2 or formate and reduction of CO2 to CH4; these studies have revealed unusual coenzymes and new biochemical reactions (1). Less understood is the pathway for conversion of acetate to CH4 and C02, which accounts for approximately two-thirds of the methane produced in most anaerobic environments. This pathway involves cleavage of acetate with transfer of the methyl group to 2-mercaptoethanesulfonic acid (HS-CoM) to form 2-(methylthio)ethanesulfonic acid (CH3-S-CoM) (2); oxidation of the carbonyl group to CO2 supplies the electron pair required for reductive demethylation of CH3-S-CoM to CH4 (3). The enzyme mechanisms underlying these reactions are unresolved. It has been postulated that CO dehydrogenase (CODH), abundant in acetate-grown methanogens, cleaves the C-C and C-S bonds of activated acetate (acetyl-CoA). Recently, acetyl-CoA synthesis and cleavage activities were reported for an enzyme complex that also contained CODH activity and was isolated from acetate-grown Methanosarcina thermophila (4, 5). In addition, an analysis of acetate auxotrophs has recently provided evidence supporting the proposed involvement of CODH in acetyl-CoA synthesis for cell carbon by autotrophic C02-reducing methanogens (6). These findings firmly support the proposed functions of CODH from methanogens and make studies on the biochemical mechanism of this enzyme necessary.CODH also catalyzes the synthesis of acetyl-CoA in the energy-yielding Wood pathway of acetogenic eubacteria (7). The archaebacteria and eubacteria are at the extremes of bacterial evolution (8); thus, it is likely that CODH is an ancient enzyme and that the various present-day CODH enzymes originated from a common ancestor. The comparative biochemistry of this enzyme f...

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The purified nickel-containing CO dehydrogenase complex isolated from methanogenic Methanosarcina thermophila grown on acetate is able to catalyze the exchange of [1-14C] acetyl-coenzyme A (CoA) (carbonyl group) with 12CO as well as the exchange of [3'-32P]CoA with acetyl-CoA. Kinetic parameters for the carbonyl exchange have been determined: Km (acetyl-CoA) = 200 ,uM, Vmax = 15 min-'. CoA is a potent inhibitor of this exchange (K1 = 25 ,uM) and is formed under the assay conditions because of a slow but detectable acetyl-CoA hydrolase activity of the enzyme. Kinetic parameters for both exchanges are compared with those previously determined for the acetyl-CoA synthase/CO dehydrogenase from the acetogenic Clostridium thermoaceticum. Collectively, these results provide evidence for the postulated role of CO dehydrogenase as the key enzyme for acetyl-CoA degradation in acetotrophic bacteria.Acetogens and methanogens possess novel pathways for the synthesis of acetyl-coenzyme A (acetyl-CoA) from single carbon units which allow these organisms to grow on CO2 as the sole carbon source (5). Other strains of methanogenic bacteria such as Methanosarcina species are able to grow on analogous Cl feedstocks including methanol and methylamine by using variations of this same pathway for carbon assimilation. The key enzyme in these cases is carbon monoxide dehydrogenase, a complex nickel enzyme so named for its ability to catalyze the reduction of CO2 to CO. However, the enzyme has been implicated in a number of cases as the catalyst for the carbon-carbon bond formation from suitable methyl donors and enzymatically generated CO from CO2 to give acetyl units.In the acetogenic Clostridium thermoaceticum, one molecule of CO2 is reduced to formate and formate is reduced to a methyl group by using tetrahydrofolate as a carrier; enzymatic transfer to a corrinoid iron/sulfur protein yields the methylated organocobalt species (19). Nickel-containing CO dehydrogenase (more appropriately called acetyl-CoA synthase) then acts as the site of carbon-carbon bond coupling by accepting the methyl group and catalyzing its carbonylation in the presence of CoA to give acetyl-CoA as product. The enzyme from C. thermoaceticum is the most well characterized acetogenic enzyme containing 2 Ni, 11 Fe, and 1 Zn atoms per active site; there is some discussion about the subunit composition of the functional complex as isolated: c433 [

The carbon monoxide dehydrogenase (CODH) complex from Methanosarcina thermophika catalyzed the synthesis of acetyl coenzyme A (acetyl-CoA) from CH3I, CO, and coenzyme A (CoA) at a rate of 65 nmol/min/mg at 550C. The reaction ended after 5 min with the synthesis of 52 nmol of acetyl-CoA per nmol of CODH complex. The optimum temperature for acetyl-CoA synthesis in the assay was between $5 and 600C; the rate of synthesis at 550C was not significantly different between pHs 5.5 and 8.0. The rate of acetyl-CoA synthesis was independent of CoA concentrations between 20 tuM and 1 mM; however, activity was inhibited 50% with 5 mM CoA. Methylcobalamin did not substitute for CH3I in acetyl-CoA synthesis; no acetyl-CoA or propionyl coenzyme A was detected when sodium acetate or CH3CH2I replaced CH3I in the assay mixture. CO could be replaced with CO2 and titanium(EI) citrate. When CO2 and "'CO were present in the assay, the specific activity of the acetyl-CoA synthesized was 87% of the specific activity of "'CO, indicating that CO was preferentially incorporated into acetyl-CoA without prior oxidation to free CO2. Greater than 100 ILM potassium cyanide was required to significantly inhibit acetyl-CoA synthesis, and 500 RM was required for 50% inhibition; in contrast, oxidation of CO by the CODH complex was inhibited 50% by approximately 10