Studies of the Mechanism of Phenol Hydroxylase: Effect of Mutation of Proline 364 to Serine † Dong Xu, ‡ Cristofer Enroth, § Ylva Lindqvist, | David P. Ballou,* ,‡ and Vincent Massey ‡,⊥ Department of Biological Chemistry, UniVersity of Michigan Medical School, Ann Arbor, Michigan 48109-0606, EMBL c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany, and Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden ReceiVed July 1, 2002; ReVised Manuscript ReceiVed September 11, 2002 ABSTRACT: An active site residue in phenol hydroxylase (PHHY), Pro364, was mutated to serine to investigate its role in enzymatic catalysis. In the presence of phenol, the reaction between the reduced flavin of P364S and oxygen is very fast, but only 13% of the flavin is utilized to hydroxylate the substrate, compared to nearly 100% for the wild-type enzyme. The oxidative half-reaction of PHHY using m-cresol as a substrate is similarly affected by the mutation. Pro364 was suggested to be important in stabilizing the transition state of the oxygen transfer step by forming a hydrogen bond between its carbonyl oxygen and the C4a-hydroperoxyflavin [Ridder, L., Mullholland, A. J., Rietjens, I. M. C. M., and Vervoort, J. (2000) J. Am. Chem. Soc. 122, 8728-8738]. The P364S mutation may weaken this interaction by increasing the flexibility of the peptide chain; hence, the transition state would be destabilized to result in a decreased level of hydroxylation of phenol. However, when the oxidative half-reaction was studied using resorcinol as a substrate, the P364S mutant form was not significantly different from the wild-type enzyme. The rate constants for all the reaction steps as well as the hydroxylation efficiency (coupling between NADPH oxidation and resorcinol consumption) are comparable to those of the wild-type enzyme. It is suggested that the function of Pro364 in catalysis, stabilization of the transition state, is not as important in the reaction with resorcinol, possibly because the position of hydroxylation is different with resorcinol than with phenol and m-cresol. Phenol hydroxylase (PHHY, 1 EC 1.14.13.7) is a flavo- protein monooxygenase from the aerobic topsoil yeast Trichosporon cutaneum (1, 2). It catalyzes a reaction that incorporates an atom from molecular oxygen into the ortho position of phenol or its simple derivatives (3-6). In the proposed mechanism of PHHY (Scheme 1), oxygen reacts with enzyme-bound FAD, which has been reduced by NADPH, to form C4a-hydroperoxyflavin (intermediate I). The flavin intermediate then undergoes nucleophilic attack by the phenolic substrate. As a result, the distal hydroxyl group of the hydroperoxyflavin is transferred to the substrate to form intermediate II, a complex of C4a-hydroxyflavin and a cyclohexadienone, the nonaromatic oxygenated product. The reaction cycle is completed by rearomatization of the product and dehydration of intermediate III (C4a-hydroxy- flavin). The structure of PHHY with phenol bound (7) reveals that Pro364 could be important in enzymatic catalysis (Figure 1A). According to quantum and molecular mechanical computations (8), the carbonyl oxygen of Pro364 forms a hydrogen bond with the hydroperoxide of intermediate I (Scheme 2, top). The partial negative charge on the oxygen atom of the residue is suggested to stabilize the hydroxyl group of intermediate I that is attacked by substrate in the transition state. This interaction is suggested to lower the energy barrier for the oxygen transfer (phenol hydroxylation) step by 3 kcal/mol (8). To test the hypothetical function of Pro364 in the catalysis of PHHY, the residue was changed by mutation to serine to allow more flexibility of the carbonyl oxygen. Kinetic and other studies of the P364S mutant enzyme show how the added flexibility affects catalysis. MATERIALS AND METHODS Mutagenesis. The plasmid (pRJ6C) that encodes the gene of phenol hydroxylase (phyA)(9) was provided by J. Reiser (National Institutes of Health, Bethesda, MD). In the plasmid, phyA is ligated to the Escherichia coli expression vector pKK223-3 pelB. The plasmid includes the tac promoter for † Financial support was received from the U.S. Public Health Service (Grants GM 20877 and GM 64711 to D.P.B. and Grant GM 11106 to V.M.). * To whom correspondence should be addressed: Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109- 0606. E-mail: dballou@umich.edu. Phone: (734) 764-9582. Fax: (734) 763-4581. ‡ University of Michigan. § EMBL c/o DESY. | Karolinska Institutet. ⊥ Deceased August 26, 2002. 1 Abbreviations: PHHY, phenol hydroxylase from T. cutaneum; PHBH, p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa; CDO, catechol 2,3-dioxygenase; SOD, superoxide dismutase; FAD, flavin adenine dinucleotide; IPTG, isopropyl 1-thio--D-galactopyrano- side; Pi, phosphate; FlHOOH, enzyme-bound flavin hydroperoxide; FlHOH, enzyme-bound flavin hydroxide; WT, wild-type enzyme; TCA, trichloroacetic acid; CT, charge transfer; 2,4-D, 2,4-dihydroxybenzoate; KIE, kinetic isotope effect. 13627 Biochemistry 2002, 41, 13627-13636 10.1021/bi020446n CCC: $22.00 © 2002 American Chemical Society Published on Web 10/26/2002