A thioester substrate binds to the enzyme Arthrobacter thioesterase in two ionization states: evidence from Raman difference spectroscopy

4‐Hydroxybenzoyl‐CoA (4‐HB‐CoA) thioesterase from Arthrobacter is the final enzyme catalyzing the hydrolysis of 4‐HB‐CoA to produce coenzyme A and 4‐hydroxybenzoic acid in the bacterial 4‐chlorobenzoate dehalogenation pathway. Using a mutation E73A that blocks catalysis, stable complexes of the enzyme and its substrate can be analyzed by Raman difference spectroscopy. Here we have used Raman difference spectroscopy, in the non‐resonance regime, to characterize 4‐HB‐CoA bound in the active site of the E73A thioesterase. In addition, we have characterized complexes of the wild‐type enzyme complexed with the unreactive substrate analog 4‐hydroxyphenacyl‐CoA (4‐HP‐CoA). Both sets of complexes show evidence for two forms of the ligand in the active site: one population has the 4‐hydroxy group protonated, 4‐OH; while the second has the group as the hydroxide, 4‐O⁻. For bound 4‐HP‐CoA, X‐ray data show that glutamate 78 is close to the 4‐OH in the complex and it is likely that this is the proton acceptor for the 4‐OH proton. Although the pK_a of the 4‐OH group on the free substrate in aqueous solution is 8.6, the relative populations of ionized and neutral 4‐HB‐CoA bound to E73A remain invariant between pH 7.3 and 9.8. The invariance with pH suggests that the 4‐OH and the ‐COO⁻ of E78 constitute a tightly coupled pair where their separate pK_a ‘s lose their individual qualities. Narrow band profiles are seen in the CO double bond and C‐S regions, suggesting that the hydrolyzable thioester group is rigidly bound in the active site in a syn gauche conformation. Copyright © 2011 John Wiley & Sons, Ltd.