Pulse-Chase Studies of the Synthesis of Acetyl-CoA by Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase: EVIDENCE FOR A RANDOM MECHANISM OF METHYL AND CARBONYL ADDITION

Javier Seravalli,Stephen W Ragsdale

doi:10.1074/jbc.m709470200

Abstract

Carbon monoxide dehydrogenase/acetyl-CoA synthase catalyzes acetyl-CoA synthesis from CO, CoA, and a methylated corrinoid iron-sulfur protein, which acts as a methyl donor. This reaction is the last step in the Wood-Ljungdahl pathway of anaerobic carbon fixation. The binding sequence for the three substrates has been debated for over a decade. Different binding orders imply different mechanisms (i.e. paramagnetic versus diamagnetic mechanisms). Ambiguity arises because CO and CoA can each undergo isotopic exchange with acetyl-CoA, suggesting that either of these two substrates could be the last to bind to the acetyl-CoA synthase active site. Furthermore, carbonylation, CoA binding, and methyl transfer can all occur in the absence of the other two substrates. Here, we report pulse-chase studies, which unambiguously establish the order in which the three substrates bind. Although a CoA pulse is substantially diluted by excess CoA in the chase, isotope recovery of a pulse of labeled CO or methyl group is unaffected by the presence of excess unlabeled CO or methyl group in the chase. These results demonstrate that CoA is the last substrate to bind and that CO and the methyl group bind randomly as the first substrate in acetyl-CoA synthesis. Up to 100% of the methyl groups and CoA and up to 60-70% of the CO employed in the pulse phase can be trapped in the product acetyl-CoA.

Highlights

There are three types of CO dehydrogenase (CODH)2 [1, 2]
The 72-kDa ␤-subunit is CODH, which catalyzes the reversible oxidation of CO to CO2 (Reaction 2) at the C-cluster, a novel [NiFe4S4–5] cluster [3, 4], whereas the 82-kDa ␣-subunit is acetyl-CoA synthase (ACS), which catalyzes the condensation of three substrates, CoA, CO, and a methyl group from the methylated corrinoid iron-sulfur protein (CH3CFeSP), to produce acetyl-CoA
In the absence of CFeSP, CODH1⁄7ACS catalyzes the CO/acetyl-CoA exchange reaction between free CO in solution and [1-14C]acetyl-CoA [22,23,24] (Reaction 4). This reaction occurs at a rate similar to that of acetyl-CoA synthesis and involves cleavage of the C–C and C–S bonds of acetyl-CoA to form a quaternary complex in which 14CO, CH3, and CoA independently bind to ACS; bound 14CO undergoes isotopic exchange with CO in solution [25,26,27]

Summary

Introduction

There are three types of CO dehydrogenase (CODH)2 [1, 2]. These include a copper molybdopterin iron-sulfur protein that is present in aerobic bacteria and an NiFeS enzyme that is present in anaerobic microbes. In the absence of CFeSP, CODH1⁄7ACS catalyzes the CO/acetyl-CoA exchange reaction between free CO in solution and [1-14C]acetyl-CoA [22,23,24] (Reaction 4) This reaction occurs at a rate similar to that of acetyl-CoA synthesis and involves cleavage of the C–C and C–S bonds of acetyl-CoA to form a quaternary complex in which 14CO, CH3, and CoA independently bind to ACS; bound 14CO undergoes isotopic exchange with CO in solution [25,26,27]. This reaction occurs with retention of stereochemical configuration at the methyl moiety of acetyl-CoA [9]. Many cycles of cleavage and resynthesis of the C–S bond of acetyl-CoA can occur for each cycle of C–C bond synthesis/cleavage, indicating that CoA is the last substrate to add and that it reacts with an acetyl-enzyme intermediate

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Conclusion