A functional Ni-Ni-[4Fe-4S] cluster in the monomeric acetyl-CoA synthase from Carboxydothermus hydrogenoformans

Abstract

In anaerobic microorganisms employing the acetyl-CoA pathway, acetyl-CoA synthase (ACS) and CO dehydrogenase (CODH) form a complex (ACS/CODH) that catalyzes the synthesis of acetyl-CoA from CO, a methyl group, and CoA. Previously, a [4Fe-4S] cubane bridged to a copper-nickel binuclear site (active site cluster A of the ACS component) was identified in the ACS Mt /CODH Mt from Moorella thermoacetica whereas another study revealed a nickel-nickel site in the open form of ACS Mt , and a zink-nickel site in the closed form. The ACS Ch of the hydrogenogenic bacterium Carboxydothermus hydrogenoformans was found to exist as an 82.2-kDa monomer as well as in a 1:1 molar complex with the 73.3-kDa CODHIII Ch . Homogenous ACS Ch and ACS Ch /CODHIII Ch catalyzed the exchange between [1- 14 C]acetyl-CoA and 12 CO with specific activities of 2.4 or 5.9 μmol of CO per min per mg, respectively, at 70°C and pH 6.0. They also catalyzed the synthesis of acetyl-CoA from CO, methylcobalamin, corrinoid iron-sulfur protein, and CoA with specific activities of 0.14 or 0.91 μmol of acetyl-CoA formed per min per mg, respectively, at 70°C and pH 7.3. The functional cluster A of ACS Ch contains a Ni-Ni-[4Fe-4S] site, in which the positions proximal and distal to the cubane are occupied by Ni ions. This result is apparent from a positive correlation of the Ni contents and negative correlations of the Cu or Zn contents with the acetyl-CoA/CO exchange activities of different preparations of monomeric ACS Ch , a 2.2-Å crystal structure of the dithionite-reduced monomer in an open conformation, and x-ray absorption spectroscopy.