Evidence for Electrophilic Catalysis in the 4-Chlorobenzoyl-CoA Dehalogenase Reaction: UV, Raman, and ¹³C-NMR Spectral Studies of Dehalogenase Complexes of Benzoyl-CoA Adducts

This paper reports on the mechanism of substrate activation by the enzyme 4-chlorobenzoyl coenzyme A dehalogenase. This enzyme catalyzes the hydrolytic dehalogenation of 4-chlorobenzoyl coenzyme A (4-CBA-CoA) to form 4-hydroxybenzoyl coenzyme A (4-HBA-CoA). The mechanism of this reaction is known to involve attack of an active site carboxylate (Asp or Glu side chain) at C(4) of the substrate benzoyl ring to form a Meisenheimer complex. Loss of chloride ion from this intermediate results in the formation of an arylated enzyme intermediate. The arylated enzyme is hydrolyzed to free enzyme plus 4-HBA-CoA by the addition of water at the acyl carbon [Yang, G., Liang, P.-H., & Dunaway- Mariano, D. (1994) Biochemistry 33, 8527]. The present studies have focused on the activation of the 4-CBA-CoA for nucleophilic attack by the active site carboxylate group. UV-visible, ¹³C-NMR, and Raman spectroscopic techniques were used to monitor changes in the distribution of the πelectrons of the benzoyl moiety of benzoyl-CoA adducts [substituted at C(4) with methyl (4-MeBA-CoA), methoxy (4-MeOBA-CoA), or hydroxyl (4-HBA-CoA) groups or at C(2) or C(3) with a hydroxyl group (2-HBACoA and 3-HBA-CoA)] resulting from the binding of these ligands to the dehalogenase active site. The UV-visible spectra measured for 4-HBA-CoA in aqueous buffer at pH 7.5 and in the dehalogenase active site revealed that a large red shift (from 292 to 373 nm) in the λ_max of the benzoyl moiety occurs upon binding. The UV-visible spectra of 4-MeBA-CoA and 4-MeOBA-CoA in aqueous buffer show λ_max values for the benzoyl chromophore at ca. 260 nm (ε = 4 mM^-1 cm^-1) and at 292 nm (ε = 11 mM^-1 cm^-1), respectively, which are shifted to 302 nm (ε = 6 mM^-1 cm^-1) and to 323 nm (ε = 10 mM^-1 cm^-1) upon enzyme complexation. In contrast, the other benzoyl-CoA adducts displayed essentially the same spectral properties on and off the enzyme. The ¹³C-NMR spectrum was measured for [¹³C=0]- 4-HBA-CoA (enriched with the carbon-13 isotope at the thioester C=0) in aqueous buffer and in the enzyme active site. A 2.8 ppm downfield shift of the ¹³C=0 resonance was observed upon binding to the enzyme. The Raman spectra of 4-HBA-CoA and 4-MeBA-CoA bound to the dehalogenase active site provide evidence for polarization of the benzoyl πelectrons. Specifically, the thioester C=0 stretch observed at 1646 cm^-1 and the in-plane phenyl C-H stretches observed at 1221 and 1173 cm^-1 in the spectrum of 4-HBA-CoA in aqueous buffer are not discernible in the spectrum of the enzyme-bound 4-HBA-CoA, and the benzene ring modes 8a and 8b observed at 1603 and 1589 cm^-1 are replaced by features at 1560 and 1525 cm^-1, respectively. The aromatic ring-stretching modes observed for 4-MeBACoA at 1609 and 1581 cm^-1 in buffer undergo shifts to 1596 and 1570 cm^-1 upon enzyme complexation, while the thioester C=0 stretch band observed at 1651 cm^-1 (buffer) is shifted to 1610 cm^-1 (enzyme). The UV-visible absorption, 13C-NMR, and Raman spectral data are interpreted as evidence for substrate activation via polarization of the benzoyl electrons away from C(4) and onto the thioester carbonyl oxygen.