Evidence for Intersubunit Communication during Acetyl-CoA Cleavage by the Multienzyme CO Dehydrogenase/Acetyl-CoA Synthase Complex from Methanosarcina thermophila

Abstract

The carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) from Methanosarcina thermophila is part of a five-subunit complex consisting of ␣, ␤, ␥, ␦, and ⑀ subunits. The multienzyme complex catalyzes the reversible oxidation of CO to CO 2 , transfer of the methyl group of acetyl-CoA to tetrahydromethanopterin (H 4 MPT), and acetyl-CoA synthesis from CO, CoA, and methyl-H 4 MPT. The ␣ and ⑀ subunits are required for CO oxidation. The ␥ and ␦ subunits constitute a corrinoid iron-sulfur protein that is involved in the transmethylation reaction. This work focuses on the ␤ subunit. The isolated ␤ subunit contains significant amounts of nickel. When proteases truncate the ␤ subunit, causing the CODH/ACS complex to dissociate, the amount of intact ␤ subunit correlates directly with the EPR signal intensity of Cluster A and the activity of the CO/acetyl-CoA exchange reaction. Our results strongly indicate that the ␤ subunit harbors Cluster A, a NiFeS cluster, that is the active site of acetyl-CoA cleavage and assembly. Although the ␤ subunit is necessary, it is not sufficient for acetyl-CoA synthesis; interactions between the CODH and the ACS subunits are required for cleavage or synthesis of the C-C bond of acetyl-CoA. We propose that these interactions include intramolecular electron transfer reactions between the CODH and ACS subunits.

Nickel in Subunit β of the Acetyl-CoA Decarbonylase/Synthase Multienzyme Complex in Methanogens

“…6, at low redox potential acetylCoA was generated over time from CoA and the one-carbon precursors CO and methylcobalamin. Previously, it was shown unequivocally that the ACDS ␤ subunit is responsible for activation of the C-S bond of acetyl-CoA (2, 3); however, only indirect evidence for activation of the C-C bond was available (2,4). The results presented here demonstrate for the first time that the ␤ subunit active site indeed has the intrinsic chemical properties needed for C-C bond activation.…”

“…When reacted with CO in the presence of a reducing agent, the isolated clostridial ␣ subunit exhibited an EPR spectrum similar to the A cluster NiFeC signal found in earlier studies on the intact ACDS complex and the native clostridial CODH/ACS enzyme (11). Characterization of the ability of the ACDS ␤ subunit to bind acetyl-CoA and CoA, and to catalyze acetyl group transfer reactions by way of a high energy acetyl-enzyme intermediate formed on the enzyme at low redox potentials (3), and other studies (4), further implicated the ␤ subunit A center as the site where acetyl C-C bond activation takes place. Functional preparations of the clostridial ␣ subunit have now been obtained that catalyze acetyl-CoA formation from CoA and CO in the presence of methylated clostridial corrinoid iron-sulfur protein (12).…”

“…The carbonyl group is oxidized to CO 2 by a process involving the ␣⑀ CO dehydrogenase subcomponent, with regeneration of the reduced form of the ␤ subunit. Previous studies on the ␤ subunit have focused on a C-terminally truncated form of the protein purified from the native ACDS complex following partial proteolytic digestion (2)(3)(4).…”

Tight Coupling of Partial Reactions in the Acetyl-CoA Decarbonylase/Synthase (ACDS) Multienzyme Complex from Methanosarcina thermophila

“…3B, the free ACDS ␤ subunit does not catalyze CO exchange at an increased rate, with the observed activity being only slightly above the level of detection, even lower than found for the ACDS complex. As shown in a previous study, loss of carbonyl exchange activity of ACDS also takes place upon disruption of the complex by partial proteolysis (10).…”

“…For example, methyl group transfer to the corrinoid protein (Scheme 1) requires a separate methyltransferase enzyme in bacteria, but the activity is intrinsic to the ␥␦ subcomponent in ACDS. Furthermore, the isotope exchange reaction (Reaction 2), [1-14 C]acetyl-CoA ϩ CO^acetyl-CoA ϩ 14 CO REACTION 2 which takes place as a consequence of C-S and C-C bond cleavage activity (26), typically exceeds the rate of overall acetyl-CoA synthesis by as much as 3-and 17-fold in experiments with CODH Mt /ACS Mt and the monomeric ACS Ch , respectively (24,27,28), but exhibits low (sometimes variable) rates with the ACDS complex (10,29). Unpublished data 3 from our laboratory suggest that ACDS catalyzes CO exchange much more slowly than the overall synthesis and cleavage of acetylCoA (Reaction 1) or any of the individual partial reactions (Scheme 1).…”