Raman Study of the Polarizing Forces Promoting Catalysis in 4-Chlorobenzoate-CoA Dehalogenase ² John Clarkson, ‡ Peter J. Tonge, § Kim L. Taylor, | Debra Dunaway-Mariano, | and Paul R. Carey* ,‡ Department of Biochemistry, Case Western ReserVe UniVersity, 10900 Euclid AVenue, CleVeland, Ohio 44106, Department of Chemistry, State UniVersity of New York at Stony Brook, Stony Brook, New York 11794, and Department of Chemistry and Biochemistry, UniVersity of Maryland, College Park, Maryland 21742 ReceiVed April 23, 1997; ReVised Manuscript ReceiVed June 12, 1997 X ABSTRACT: The enzyme 4-chlorobenzoate-CoA dehalogenase catalyzes the hydrolysis of 4-chlorobenzoate- CoA (4-CBA-CoA) to 4-hydroxybenzoyl-CoA (4-HBA-CoA). In order to facilitate electrophilic catalysis, the dehalogenase utilizes a strong polarizing interaction between the active site residues and the benzoyl portion of the substrate [Taylor, K. L., et al. (1995) Biochemistry 34, 13881]. As a result of this interaction, the normal modes of the benzoyl moiety of the bound 4-HBA-CoA undergo a drastic rearrangement as shown by Raman spectroscopy. Here, we present Raman difference spectroscopic data on the product- enzyme complex where the product’s benzoyl carbonyl is labeled with 18 O (Cd 18 O) or 13 C( 13 CdO) or where the 4-OH group is labeled with 18 O. The data demonstrate that the carbonyl group participates in the most intense normal modes occurring in the Raman spectrum in the 1520-1560 cm - 1 region. The substrate analog 4-methylbenzoate-CoA (4-MeBA-CoA) has also been characterized by Raman difference spectroscopy in its free form and bound to the dehalogenase. Upon binding, the 4-MeBA-CoA shows evidence of polarization within the delocalized π-electrons, but to a lesser extent compared to that seen for the product. The use of 4-MeBA-CoA labeled with 18 O at the carbonyl enables us to estimate the degree of electron polarization within the CdO group of the bound 4-MeBA-CoA. The CdO stretching frequency occurs near 1663 cm -1 in non-hydrogen bonding solvents such as CCl 4 , near 1650 cm -1 in aqueous solution, and near 1610 cm -1 in the active site of dehalogenase. From model studies, we can estimate that in the active site the carbonyl group behaves as though it is being polarized by hydrogen bonds approximately 57 kJ mol -1 in strength. Major contributions to this polarization come from hydrogen bonds from the peptide NHs of Gly114 and Phe64. However, an additional contribution, which may account for up to half of the observed shift in ν CdO , originates in the electrostatic field due to the R-helix dipole from residues 121-114. The helix which terminates at Gly114, near the CdO group of the bound benzoyl, provides a dipolar electrostatic component which contributes to the polarization of the CdO bond and to the polarization of the entire benzoyl moiety. The effect of both the helix dipole and the hydrogen bonds on the CdO is a “pull” of electrons onto the carbonyl oxygen, which, in turn, polarizes the electron distribution within the benzoyl π-electron system. The ability of these two factors to polarize the electrons within the benzoyl moiety is increased by the environment about the benzoyl ring; it is surrounded by hydrophobic residues which provide a low-dielectric constant microenvironment. Electron polarization promotes catalysis by reducing electron density at the C4 position of the benzoyl ring, thereby assisting attack by the side chain of Asp145. An FTIR study on the model compound 4-methylbenzoyl S-ethyl thioester, binding to a number of hydrogen bonding donors in CCl 4 , is described and is used to relate the observed shift of the CdO stretching mode of 4-MeBA-CoA in the active site to the hydrogen bonding strength value. Since the shift of the CdO frequency upon binding is due to hydrogen bonding and helix dipole effects, we refer to this bonding strength as the effective hydrogen bonding strength. 4-Chlorobenzoate-coenzyme A dehalogenase catalyzes the hydrolytic dehalogenation of 4-chlorobenzoate-coenzyme A (4-CBA-CoA) to 4-hydroxybenzoate-CoA (4-HBA-CoA). This enzyme has been discovered in a number of soil- dwelling bacterial strains where it functions along with 4-CBA-CoA ligase and 4-HBA-CoA thioesterase to convert p-chlorobenzoic acid to p-hydroxybenzoic acid (for a review, see ref 1). Studies of the Pseudomous sp. strain CBS3 4-CBA-CoA dehalogenase have shown that it utilizes a unique form of catalysis in which an active site carboxylate bonds to C4 of the benzoyl ring of the bound substrate to form a Meisenheimer complex which then proceeds to product through chloride ion expulsion, the formation of an arylated intermediate, and hydrolysis as seen in Scheme 1 (2, 3). The nature of the active site forces which increase the electrophilic character of the C4 position is of interest since the formation of a Meisenheimer-like complex in the reaction is not chemically facile (4, 5). An indication that strong electrostatic forces are present in the active site came from ² This work was supported by NIH Grants GM-36360 (to D.D.-M.) and GM-54072 (to P.R.C.). * Author to whom correspondence should be addressed. E-mail: carey@biochemistry.cwru.edu. Fax: (216) 368-4544. ‡ Case Western Reserve University. § State University of New York at Stony Brook. | University of Maryland. X Abstract published in AdVance ACS Abstracts, August 1, 1997. 10192 Biochemistry 1997, 36, 10192-10199 S0006-2960(97)00941-0 CCC: $14.00 © 1997 American Chemical Society