Synthesis of acetyl coenzyme A by carbon monoxide dehydrogenase complex from acetate-grown Methanosarcina thermophila

The carbon monoxide dehydrogenase (CODH) complex from Methanosarcina hermophila 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 55°C. 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 55 and 60°C; the rate of synthesis at 55°C was not significantly different between pHs 5.5 and 8.0. The rate of acetyl-CoA synthesis was independent of CoA concentrations between 20 μM 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(III) citrate. When CO₂ and ¹⁴CO were present in the assay, the specific activity of the acetyl-CoA synthesized was 87% of the specific activity of ¹⁴CO, indicating that CO was preferentially incorporated into acetyl-CoA without prior oxidation to free CO₂. Greater than 100 μM potassium cyanide was required to significantly inhibit acetyl-CoA synthesis, and 500 μM was required for 50% inhibition; in contrast, oxidation of CO by the CODH complex was inhibited 50% by approximately 10 μM potassium cyanide.