# From two to one: resolving CO binding in acetyl-CoA synthase

## TL;DR

This study shows that acetyl-CoA synthase binds only one CO molecule under normal conditions, resolving a long-standing question about its CO coordination.

## Contribution

The study provides the first direct evidence that acetyl-CoA synthase binds a single CO ligand under native conditions.

## Key findings

- ACS binds a single CO ligand under ambient, near-native conditions.
- Advanced IR spectroscopy and computational methods reveal the true CO coordination in ACS.
- The two-CO model is not physiologically relevant under native conditions.

## Abstract

Acetyl-CoA synthase (ACS) catalyzes the condensation of acetyl-CoA from carbon monoxide (CO), a methyl group, and coenzyme A, enabling the fixation of CO into biomolecules. Recent low-temperature ENDOR studies proposed that the enzyme can bind two CO ligands in its reduced Ared–CO state, reshaping the view of CO coordination and inhibition of ACS. However, whether this two-CO model reflects a physiologically relevant state has remained an open question. To address this issue, we examined ACS under near-native, ambient conditions using ultrafast and two-dimensional infrared spectroscopy, complemented by anharmonic frequency calculations. These methods provide a wealth of structural and dynamical information beyond insights from conventional IR absorption spectroscopy, allowing a direct view of CO coordination in the Ared–CO state. Our results demonstrate that ACS binds a single CO ligand under ambient conditions. This finding clarifies the stoichiometry of CO coordination in ACS and underscores the broader potential of advanced IR spectroscopy, combined with computation, to unravel ligand binding in complex bioorganometallic systems.

Acetyl-CoA synthase is a key enzyme in biological carbon fixation. Combining ultrafast and two-dimensional infrared spectroscopy with anharmonic frequency calculations, we demonstrate that the enzyme binds a single CO ligand under native conditions.